Eldering, Economics, and Calling to Ministry [9Marks Blog: Building Healthy Churches] 2013-10-31 08:33
In my first post in this series, I suggested that saying you’re “called” to ministry carries a double presumption. You’re saying you think you are, or soon will be, (1) qualified to be an elder and (2) sufficiently gifted in ministry that a church should pay you to do it. “Calling” language might imply more, but it cannot mean less.
You could call these two ingredients “eldering” and “economics”: you want to be an elder, and you want being an elder to be your job. In this article I’m going to drill into those two ideas in the hope of helping us think more wisely and biblically about “calling” to ministry.
ELDERING
Some of you might be wondering, what’s an elder? And what does that have to do with calling to ministry?
“Elder” is simply a more common biblical title for the office we typically call “pastor” (e.g., Acts 20:17, 1 Tim. 5:17, Tit. 1:5, James 5:14). In Acts 20:28 Paul tells the elders of the church in Ephesus to care for the church in which the Holy Spirit has made them overseers. The basic meaning of that verb “care for” (poimaino) is “to shepherd.” And our English word pastor comes from the Greek word for “shepherd.” So, Paul is telling the elders to shepherd the flock; that is, to pastor the church. What do elders do? They pastor.
Peter makes the same connection with the same word when he exhorts elders: “shepherd the flock of God that is among you” (1 Pet. 5:1-2). The office is elder, the work is pastoring. Every elder is a pastor.
This pastoral office is also referred to as an overseer (Acts 20:28, Phil. 1:1, 1 Tim. 3:1, Tit. 1:7) and, in one case, a pastor-teacher (Eph. 4:11). The terms elder, overseer, and pastor are interchangeable. They all refer to a church’s spiritual leaders: the men who teach the word and direct the affairs of the church.
I’ll say it again: to be an elder is to be a pastor, and every pastor is an elder. What might surprise some is that Scripture never uses the term “call” or “calling” in relation to pastoral ministry. What Paul does say is this: “The saying is trustworthy: If anyone aspires to the office of overseer, he desires a noble task” (1 Tim. 3:1). Paul speaks here of desire. He speaks of aspiring to a certain office because you desire to do the work.
If you say you’re called to ministry, I don’t immediately know what you mean, or if what you mean is biblical. But if you say, “I aspire to be an elder,” I know just what you’re talking about. You desire to teach the word and shepherd God’s flock, so you desire to fulfill the office God has established for that purpose. And the apostle Paul says you desire a good thing.
ECONOMICS
But “calling” typically implies more than an office; it also implies a job. When people talk about being called to ministry, they typically don’t mean they want to be a pastor in addition to having another full-time job. Instead they mean they want pastoring to be their full-time job.
Scripture teaches that “those who proclaim the gospel should get their living by the gospel” (1 Cor. 9:14); and, “Let the one who is taught the word share all good things with the one who teaches” (Gal. 6:6). And with explicit reference to elders, Paul says, “Let the elders who rule well be considered worthy of double honor, especially those who labor in preaching and teaching. For the Scripture says, ‘You shall not muzzle an ox when it treads out the grain,’ and, ‘The laborer deserves his wages’” (1 Tim. 5:17-18). So, when possible, churches should pay their pastors.
Notice that in all these cases, financial support is tied to preaching and teaching. All elders must be able to teach (1 Tim. 3:2), and their job description includes exhorting in sound doctrine and refuting those who oppose it (Tit. 1:9). But 1 Timothy 5:17-18 seems to indicate that some elders will be especially devoted to, and gifted in, the work of preaching and teaching. These are singled out for special honor—including pay.
It’s easy to understand why. Preaching the Word is both time-consuming and essential to the life of the church. Paul told Timothy to study to show himself approved, a workman who rightly handled the word (2 Tim. 2:15). Faithful preaching takes painstaking preparation. And without faithful preaching, a congregation withers.
So, if you think of yourself as called to ministry, here’s another biblical way to specify what you mean: “I aspire to serve as an elder. Specifically, I desire to devote myself as completely as possible to the work of preaching and teaching.”
I’m not saying that preaching is the only valid full-time ministry. What I am saying is that aiming first at the office of elder, and secondly at being especially devoted to teaching the word, is a biblical goal. Since the Bible doesn’t use “calling” in this context, “calling” isn’t tethered to Scripture. Aspiration to eldership is.
A WRINKLE
But there’s a wrinkle in this fabric: not all elders are paid, so not all elders serve full-time. In fact, Paul commended his own example of bi-vocational ministry to the Ephesian elders:
You yourselves know that these hands ministered to my necessities and to those who were with me. In all things I have shown you that by working hard in this way we must help the weak and remember the words of the Lord Jesus, how he himself said, “It is more blessed to give than to receive.” (Acts 20:34-35)
Paul encouraged these elders to follow his own example of serving others not just by pastoring, but working to provide for their own and others’ material needs. There’s no intrinsic conflict between the two, nor do you need to choose between them. Being a pastor is not more spiritual or more acceptable to God than being a plumber. And a plumber who’s also a pastor is walking in Paul’s own footsteps.
So not all elders are paid to elder, which means that you might never be. Anyone who aspires to pastor should not only be reconciled to this, but embrace it. If you desire the work of an overseer, you should be willing to do it whether anyone pays you or not.
This also means you should be willing to do the work whether or not anyone ever gives you the title. Of course some of elders’ work is restricted to elders. But if you aspire to elder, you should be developing an increasingly elder-like ministry. Encourage others with the word. Help them grow in Christ-likeness. Live wisely and give wise counsel. Pray fervently for your fellow church members. If you want to lead, go lead something. Start with a family (1 Tim. 3:4-5). The best way to tell if a man should be an elder is if he already ministers like one.
Again, you aren’t a pastor until a church calls you to be their pastor. Whether you ever serve as an elder, and whether you are ever paid to serve as an elder, is up to a church to decide. Ultimately, it’s out of your hands. This should be both humbling and freeing: humbling because you shouldn’t presume on it, freeing because you can simply serve the church and trust God to open whatever doors he wants to.
GENTLE COURSE CORRECTIONS
How does this eldering-and-economics view of “calling” inform our thinking and speaking on the subject? Here I’ll suggest some gentle course corrections. (In the next post I’ll offer a few more pointed exhortations and encouragements.)
Basically, I’m recommending a way to translate “calling” into a more biblical idiom. If you say you’re called to pastoral ministry, what I hope you mean is that you aspire to be an elder, and that you aspire to devote yourself to preaching and teaching.
There are other valid ways to serve a church full-time: counseling, administration, and so on. And there are plenty of other ministry roles in and out of the church that require full-time effort but don’t pay for themselves. But for those who feel “called to the ministry,” I’d encourage you to think in terms of this twofold aspiration.
And I might encourage you not just to think this way, but also to speak this way. At least add it to your lexicon. To say “I’m called” implies you’re passive in the matter: God has done it. It therefore implies a kind of certainty that becomes almost a sin to doubt. “I’ve been questioning my calling lately,” says the seminary student. Well, maybe you should be!
On the other hand, saying you aspire to be an elder, and to serve as one full-time, acknowledges you’re not there yet. And you’re confessing that it’s in God’s and the church’s hands to determine if you ever will be. Speaking this way won’t guarantee that you hold this aspiration humbly and you’re willing to be redirected, but it does set you on the right track.
I’m not saying these two ways of speaking are utterly contradictory or that no one should ever claim a sense of God leading them to do something. But I am saying that aspiring to the office of elder is a thoroughly biblical aim. Not only that, a noble one.
Bobby Jamieson is assistant editor for 9Marks, a member of Third Avenue Baptist Church in Louisville, Kentucky, and the author of Sound Doctrine: How a Church Grows in the Love and Holiness of God (Crossway, 2013). You can follow him on Twitter.
The Double Presumption of Calling to Ministry [9Marks Blog: Building Healthy Churches] 2013-10-30 08:09
What does it mean to say that you’re “called” to pastoral ministry?
It might mean that you like the idea of getting paid to study and teach the Bible. It might mean you think going into ministry is what mature Christians do, and you want to be a mature Christian, so you want to go into ministry.
I think the common language of “calling” to ministry is slippery and possibly misleading. I don’t think it’s necessarily sinful or unbiblical. But I do think it can obscure some important issues that men aspiring to ministry, and the churches that encourage and assess them, should pay more attention to.
So in this series of posts I’m going to dig into a few issues related to calling to ministry that we sometimes neglect. The first is that calling language carries, and often conceals, a double presumption.
AHEM
Before I get to my main point, though, here’s a little throat-clearing that should suffice for the series. This series of posts is geared toward men considering or pursuing pastoral ministry. It’s also geared toward the churches which encourage and assess them—which, in one way or another, is every church.
I’ll focus on calling to vocational, pastoral ministry because it’s probably relevant to the most people. Much of what I’ll say will be relevant to aspiring missionaries and other Christian workers, but you’ll have to tweak the details yourself.
I write as one who’s currently walking this path, not as one who’s arrived. In one way or another I’ve been training for pastoral ministry for the past eight years, but I’m not yet a pastor, much less an experienced one. But as a recent seminary grad I’m surrounded by men who are working through the same issues. So consider this a view from the middle of the pack, not the finish line.
CARRYING TWINS
Calling language typically refers to a sense of divine guidance: “I think God is calling me to do this.” But whether we mean it to or not, it also has first-person implications. To say that you’re called to ministry is to say that you desire to be a pastor. It’s also to say that you think you should be a pastor—as opposed to being a plumber or painter or school principal. Which means you think you’re qualified to be a pastor.
More specifically, saying you’re called to ministry presumes you think these two things about yourself: (1) you are, or soon will be, qualified to be an elder; (2) you are, or soon will be, sufficiently gifted in ministry that a church should pay you to do it.
Unless you’re talking nonsense, claiming a divine “call” has to imply both of these things—and it can’t contradict either. But this reveals one potential problem with calling language right off the bat: if I say I’m called, who are you to contradict?
I trust that godly, humble people who use calling language would never dream of using it as a trump card against criticism or questions. But the problem is, not everyone who claims to be called to ministry is godly or humble. And the language itself encourages us to view the matter as almost a private revelation, rather than a personal desire subject to public scrutiny.
But let’s table the issues of language and guidance. If you think of yourself as called to ministry, have you taken stock of these twins the claim is carrying?
Do you think that you are, or soon will be, qualified to be an elder? Have you carefully studied the qualifications in 1 Timothy 3:1-7 and Titus 1:5-9? Have you prayerfully pursued them? Have you asked your pastors what they think of your fitness for the office, and where they see areas of needed growth?
Do you think that you are, or soon will be, sufficiently gifted in ministry that a church should pay you to do it? What in your ministry track record makes you think that? Have your own pastors encouraged this? Has any church ever offered to pay you to do ministry?
FEELS AWKWARD? IT SHOULD
My point in raising these questions is not—I repeat, not—that you need a perfect score on them before you should take any practical steps toward vocational ministry. Yet the answers to these questions, and others like them, can help determine whether your presumption is justified.
When you talk about calling to ministry you are inescapably talking about what you think about yourself. If that feels a little awkward, it should. It should give you pause. It should turn your attention to the qualifications of the office and the demands of the job.
It should also cause you to seek counsel, not just from friends and family, but from your church, especially your church’s leaders. Don’t be content with vague approval and happy wishes. Ask your church leaders tough questions about your strengths and weaknesses, your gifts and blind spots. Ask them if, in principle, they would ever hire you—and if not, why not?
Viewing “calling” as entailing a double presumption doesn’t just mean putting on the brakes—though many of us probably should. It also means that if you’ve got a strong ministry track record, godly counselors in your corner, and deep, meaningful affirmation from a church or churches, you should have a greater confidence in pursuing vocational ministry. If your own humility causes you to question your gifts and qualification, take your church’s affirmation seriously—again, provided the affirmation itself is serious. To brothers whose gifts and character are evident yet who’ve not yet found an open door for full-time ministry, I’d simply say, press on and be patient.
THREE MORE TAKEAWAYS
I’ll conclude with three more takeaways. The first is that you should view all ministry, especially vocational ministry, as a gift, not a given. Every pastoral opportunity you receive—from teaching Sunday School to a full-time pastorate—is a gift from God and his church. Treat ministry as a privilege, not an entitlement.
Second, submit your desires to the Lord. You can’t be absolutely certain that you’re called to ministry until a church calls you to be their minister. So if you think God’s the one who gave you these desires, continually give them back to him, to do with them as he pleases.
Be willing to serve him however he sees fit. Embrace off-stage service as much as the spotlight. Be as eager to help in the nursery as you are in the pulpit. I know more than one pastor whose first years of ministry included cleaning their church’s toilets. So if your desire is to serve the Lord, serve him now in whatever ways he puts before you.
Third, submit your desires to the church. If you’re not sure if you should head into ministry, enlist your church’s help, and submit to that help. Be willing to slow if they flash a yellow light, and stop if it’s red. The best preparation for handling authority well is submitting to it gladly.
It’s impossible to speak to every circumstance, but if you’re sorting through these issues I hope at least something in here gets your wheels spinning. In my next post I’ll suggest some ways eldering and economics should reframe how we think about calling to ministry.
Bobby Jamieson is assistant editor for 9Marks, a member of Third Avenue Baptist Church in Louisville, Kentucky, and the author of Sound Doctrine: How a Church Grows in the Love and Holiness of God (Crossway, 2013). You can follow him on Twitter.
Book Review: The Bruised Reed, by Richard Sibbes [9Marks Blog: Building Healthy Churches] 2013-10-29 13:29
In his book The Bruised Reed and Smoking Flax, the Puritan preacher Richard Sibbes provides a tenderhearted, Christ-exalting exposition and application of Isaiah 42:1-3. Since its initial publication in 1630, The Bruised Reed has been a source of encouragement to dejected sinners and struggling saints alike.
Sibbes follows Matthew’s interpretation of this text, seeing it to be fulfilled in the life and ministry of Jesus Christ (Matt. 12:18-20). His exposition breaks down into three basic parts: (1) Christ will not break the bruised reed; (2) Christ will not quench the smoking flax; (3) Christ will not do either of these things until he has sent forth judgment into victory. Sibbes explains the main text under these three headings and then intersperses searching application throughout the book.
Why should the busy pastor spend time reading a book written by a preacher in London nearly four centuries ago? Well, Sibbes did know a thing or two about preaching. He regularly wrote out his sermons and at the time of his death in 1635 he left over two million words on paper. The Bruised Reed is still considered to be a classic of Puritan devotion. Sibbes became known as the “heavenly Doctor Sibbes,” due in part to his God-honoring preaching and God-fearing manner of life. A couplet was written about Sibbes after his death that captures this well: “Of that good man let this high praise be given: Heaven was in him before he was in heaven.”
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Book Review: Worship Leaders, We Are Not Rock Stars, by Stephen Miller [9Marks Blog: Building Healthy Churches] 2013-10-28 11:55
Imagine yourself entering a Christian church for the first time. You’re from a different religious background, curious about the teachings of Jesus. One of the first questions you might have to ask is: “Who is the guitar-wielding, distressed jeans-wearing, facial hair-sporting figure that appears before me on a larger-than-life Jumbotron, shrouded in stage fog and professional lighting, inviting me to sing along as he croons into a microphone?” Welcome to Christianity. You have just met one of our most beloved (and caricatured!) personalities: the worship leader.
It is these personalities that Stephen Miller addresses in his thoughtful appeal, Worship Leaders, We Are Not Rock Stars. Miller writes not as a dispassionate critic but as a concerned practitioner. He serves as the worship pastor at The Journey in St. Louis, Missouri.
DIAGNOSIS AND SOLUTION
What’s the problem among those who lead corporate worship in today’s churches? According to Miller, it’s the “prideful pursuit of platform and prominence” (17). He astutely observes that such a craving for self-fulfillment is not stylistically bound, but can take place among rockers and traditional choir-directors alike (16).
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The Gospel for a Gay Friend [9Marks Blog: Building Healthy Churches] 2013-10-24 11:52

Josh had always known he was different. From his earliest memories, he looked at some boys as more than just peers. His parents knew he was “special” but they loved him for it. He learned to wear a mask and play the part of a “normal” kid until he graduated high school.
In college, Josh decided it was time to be who he really was. He made friends with other gay people and set out on sexual explorations. Josh found a refuge in his gay community and developed bonds that ran much deeper than sexual flings. Though his parents distanced themselves and old friends turned a cold shoulder, Josh felt that he was finally free in his new identity as a gay man.
Josh is no caricature. His experiences and story are true, and they are common.
What if Josh were your neighbor or your co-worker or your son? How would you give the gospel to him? How would you tell him about the forgiveness of sins, the community of believers, and true identity in Jesus?
In one sense, we would assume there is no real difference in the way we’d give Josh the good news compared to any other person. Just because Josh is sexually attracted to people of the same gender doesn’t make him foundationally different from anyone else.
For many of my Christian friends who love Jesus and struggle with same-sex attraction, the beauty of the gospel is that it addresses every area of their life, not just one expression of the fall. All of us who are believers know this. Whether we were once atheists, liars, Muslims, or self-righteous church attenders, there is no magical gospel just for “our sin.” At the foot of the cross we are all equally in need of God’s amazing grace.
At the same time, Josh has very real questions that need to be answered. In the same way an atheist, Muslim or self-righteous person would need the gospel to address them personally, we should learn to love Josh where he is in his consideration of Jesus’ claims. He has real questions that he struggles with, and we should seek to help him find those answers.
IDEAS TO KEEP IN MIND
To share the gospel with Josh, or anyone who might have questions like his, here are a few ideas to keep in mind.
1. Hope in Jesus’ power to help you.
Hope in Jesus’ power to help you. It can be intimidating for people who have never struggled with same-sex attraction to share the gospel with a gay man or woman. As with anyone we share the gospel with, we fear how they may perceive us, and we may be tempted to think they would never listen. The fear of man is a snare (Prov. 29:25). So rather than getting hung up on it, we must hope in Jesus’ strength in us, not in our adequacy to bring the message (John 15:5; 2 Cor. 3:5). We must drink deeply of the gospel as we share it, because there we find the power we need to be Jesus’ witnesses (Acts 1:8). Hope in Jesus’ power to help you.
2. Hold Jesus as supreme.
Hold Jesus as supreme. Friends like Josh will often want to bring the question of sexuality to the foreground in your conversation. At the same time, we want to keep Jesus and his gospel central.
To help with this, I encourage you to ask your friend to share their story with you. Ask them to help you understand how being gay became a central part of their identity. Or, if that’s not their experience, ask them about where they do find their identity. Ask them if there have been any hard times with their journey. Part of loving people is getting to know them.
As you do this, ask them if you can tell them why you view your identity in Christ as supreme. In the end, we aren’t trying to make people straight, we want people to be saved. We never want to minimize sins that keep people from God, but at the same time we want to magnify who brings us to God. Jesus came for sinners of all kinds, and we must keep that message central.
It is also good to keep in mind that all people are sexual sinners—some in small ways, some in greater ways. This helps us to reframe the conversation from being “You’re sexually broken, you need to be like us” to “We’re all sexual sinners who need Jesus.” Jesus is the hope for all of us, no matter how the fall shows itself in our lives.
3. Have Jesus-like compassion and conviction.
Have Jesus-like compassion and conviction. Christians have sinned in at least two major ways when it comes to reaching those in the gay community. On the one hand, some have laid aside God’s clear teaching that homosexuality is a sin in an attempt to show the love of God. Love that is stripped of truth is not love but deceit. This is a grave sin against both God and man.
Have Jesus-like conviction and speak the truth in love. Share what the Bible teaches about homosexual activity (Mk. 7:21; Rom. 1:24-27; 1 Cor. 6:9-10; 1 Tim. 1:10). Share that there is a terrible judgment for those who reject Christ (Rev. 20:11-15). Share that there is a great cost in following Christ and also a great hope of forgiveness and freedom for those who do (Mk. 10:28-30). Speak the truth in love.
On the other hand, some have neglected compassion and have harbored a condescending attitude toward people who practice homosexual sin. Love that is stripped of compassion is not love but hypocrisy. This too is a grave sin because it is unlike Christ’s love toward us.
Jesus, the God-man, was unlike the world of sinners who surrounded him, yet he had compassion on them (Matt. 9:36). As we reach out to those in gay community, we must strive to do so with a similar heart. What could be more heartbreaking than for a person made in God’s image to be lost in their sin and forever separated from the love of God? Ask God to help you to see those in the gay community as he does so you can minister with conviction and compassion.
4. Have Jesus’ church be central.
Have Jesus’ church be central. As it was for Josh, the gay community is a refuge from the rejection and inner turmoil that many gay people experience. Because of this, they find a place where they are accepted in their sin and embraced for who they are.
I suspect that one of the great antidotes for this powerful tool of the evil one is the community of the church. This may seem odd in light of the way many demonize the church because of its “bigotry,” but I trust that as we build relationships with gay friends and invite them into our homes and into our lives, they will see the true community of which they have only dreamed.
This is only enhanced when we as the church grow in giving grace to our brothers and sisters in Christ who struggle with same-sex attraction. One of the most instructive times I’ve had in the past decade was when a new believer was being baptized and he shared openly about coming out of a gay life style. In his testimony he described how the church had not only shared the gospel compassionately, but also was helping him now to live as a man with the struggles of his old desires. He said that in the church, he found a refuge that challenged him not to embrace his sin, but to embrace the Savior.
Jesus said that all people will know we are his disciples by our love (John 13:34-35). As you build relationships with gay friends, invite them into your life that they may hear the gospel, but also see it portrayed through the life of your local church.
5. Help answer their questions.
Help answer their questions. There are always objections to the gospel and few of us ever feel “fully ready” to answer those objections. But God calls us to make a defense for our hope in Jesus (1 Pt. 3:15). This means we should help people wrestle with very real questions. Here are a few Josh has asked.
Part of our calling as Jesus’ ambassadors is to help people work through questions like these and see that God’s Word does have answers. If you don’t know the answer, don’t be afraid to say, “That’s a really important question, can we find the answer together?”
6. Have patience.
Have patience with them. Take the long view in evangelism. It is rare that you share the gospel with someone and they repent in the moment. That can happen, but normally the process is much longer.
Enter into evangelistic relationships for the long haul. We are an impatient people, which can tempt us to give up quickly when we don’t see results. People are people, not projects. Often, we won’t see what God is doing in their lives. View yourself as part of God’s means to help them see and hear the gospel of Jesus. Love is patient. Show them love by being in it for the long haul.
7. Hope in Jesus’ power to save.
Hope in Jesus’ power to save them. The gospel is the power of God for salvation (Rom. 1:16-17). That means that the gospel for a gay man or woman is the same gospel for a straight man or woman. Homosexuality isn’t the chief sin, unbelief is. Jesus died for all types of sins for all types of sinners.
So do not doubt the power of Christ but pray fervently for soft hearts, open doors, and lasting fruit. Trust in God’s wisdom and God’s power, not in your own. Remember that every Christian is a living miracle. If Jesus can save you, he can save anyone, including Josh.
Garrett Kell is lead pastor of Del Ray Baptist Church in Alexandria, Virginia.
Evangelism without an Altar Call [9Marks Blog: Building Healthy Churches] 2013-10-23 11:42

Five and a half years ago I preached my first sermon as the pastor of Mount Vernon Baptist Church. The minister of music stopped me before the service with a question. He wanted to know how I’d be making the altar call.
I was confused. Prior to this Sunday morning I’d been at MVBC three times, and not once did I see anyone give an altar call. I assumed the church had decided long ago to abandon the practice. I was wrong.
As it turns out, my church has a long history of closing the service with an appeal to walk the aisle in order to join the church, recommit one’s life to the Lord, or make a public profession of faith. The three Sundays I had attended were exceptions to the rule! In fact, many of the members had come to see the altar call as the primary means the church used to reach the lost. They saw the altar call as synonymous with evangelism.
WHY NOT GIVE AN ALTAR CALL?
I trust that many who give altar calls have the best of intentions. In the early nineties I attended a church whose pastor ended the service by asking every person in the congregation to close their eyes and bow their head. Next he would invite anyone who wanted to receive Christ to raise their hand and look toward the pulpit. For about thirty seconds the pastor would scan around the hall, notice the raised hands, and in a calm, soothing voice say, “Yes, brother, I see you. Good, sister, amen,” and so on. I believe this pastor meant the best for these seekers.
Nonetheless, I’m convinced that the altar call does more harm than good. The practice of granting people immediate assurance of salvation—without taking the time to test the credibility of their profession—seems unwise at best and scandalous at worst. It is unwise because the pastor cannot sufficiently know the person he’s about to affirm is a Christian. It is scandalous because it replaces the difficult and narrow gate designed by our Savior (Mark 8:34; Matt. 7:14) with an easy and wide gate designed by us. With the best of intentions, practitioners of the altar call have given many unsaved persons the false confidence that they truly know Jesus.[1]
But that’s not all. The altar call has a tendency to put the congregation’s focus in the wrong place. After the Word is preached, members and visitors alike should be examining their own hearts. Everyone should be giving serious attention to how the message calls him or her to respond. But the altar call, ironically, tends to produce the opposite response. Instead of self-examination it leads to audience-examination. People are looking around, wondering who’s going to go forward. And if no one moves, one wonders, did the pastor fail? Or worse, did God take the day off?
These are just a few reasons why I think it’s unwise to use the altar call for evangelism.
HOW TO EVANGELIZE WITHOUT AN ALTAR CALL
How should a pastor who rejects the altar call think about evangelism in a public worship service? Put another way, what does it look like for a corporate worship service to be marked by evangelistic zeal? Here are seven answers I strive for in the services I lead:
1. Be earnest.
Be earnest. Though there is nothing more important for a preacher than fidelity to gospel truth, earnestness must be a close second. God uses men whose hearts are gripped by the tragedy of sin and the reality of salvation. Until the doctrine of God’s amazing grace has settled in a preacher’s bones, it will never flash from his lips.
2. Be clear about the gospel.
Be clear about the gospel. Every passage of Scripture is a gospel text. In all of Esther, the name of God is never mentioned, and yet his handiwork is on every page. A pastor who wants to see sinners saved will faithfully teach the Bible, showing his congregation how the person and work of Christ is the point of every text.
3. Call people to repent and believe.
Call people to repent and believe. There is a place in every sermon for a pastor to invite sinners to find hope in Christ. So often I hear sermons that end with a call to stewardship, a call to risk, a call to faithfulness—but not once a call to Christ. The preacher should carefully and passionately urge his listeners to repent and believe the good news, to submit their lives to Christ the King.
4. Create space for follow-up conversations.
Create space for follow-up conversations. When I preach the gospel during my sermons, I want unbelievers to know that I’m eager to talk more about the faith I’ve just shared. Therefore I make myself available after the service to talk about the gospel and its implications.
Other pastors that I’ve talked to invite seekers to a special room after the service for prayer or conversation. Spurgeon gave over every Tuesday afternoon to counsel seekers and new believers.[2] However you decide to do it, provide opportunities for people to talk more personally about what you just preached.
5. Offer evangelistic studies.
Offer evangelistic studies. I commonly let seekers know that they are invited to join a short, straightforward study that explains the basics of the Christian faith. The study I use is Christianity Explained, a six-week study through the Gospel of Mark published by the Good Book Company. I’ve found it to be an invaluable introduction to the gospel. In fact, training in how to lead this study has become a staple class at my church.
6. Make a big deal out of baptisms.
Make a big deal out of baptisms. Of course, baptisms already are a big deal. We should recognize that each baptism is an opportunity to show the congregation that God is at work building his church.
At Mount Vernon, we ask each baptismal candidate to share his testimony to the congregation. I’ve never required this, but I’ve yet to have a person turn me down. These new Christians are eager to testify to God’s grace, and seekers are led to question their own response to the gospel.
7. Pray.
Finally, pray. In the pastoral prayer and even the closing prayer, I regularly pray that seekers would repent and believe the gospel. I pray they would submit their lives to Christ, overcoming whatever obstacles they perceive to be standing in their way. I pray that God would make himself known by drawing sinners to himself this very day.
As you can tell, I don’t give an altar call at the church I serve. But I plead every Sunday for sinners to come to Christ. Let us long to see the saints in our congregations encouraged by the gospel, and the seekers convinced of their need to repent and believe God’s good news.
Aaron Menikoff is the senior pastor of Mount Vernon Baptist Church in Sandy Springs, Georgia.
[1] For a detailed treatment of the dangers of the altar call read Erroll Hulse, The Great Invitation: Examining the Use of the Altar Call in Evangelism (Audoban Press, 2006) and D. Martyn Lloyd-Jones, Preaching & Preachers (Zondervan, 2011), chapter 14.
[2] Arnold Dallimore, Spurgeon: A New Biography (Banner of Truth, 1985), 80.
Evangelizing the Nations at Home [9Marks Blog: Building Healthy Churches] 2013-10-22 12:48
Would you be surprised if told you that Americas are increasingly inhospitable to international visitors, and that Christians can thank God for that? One university reports that 80 percent of their international students never see the inside of a local U. S. home. Longer-term immigrants seem to fare little better.
So why can Christians thank God? If unbelievers have lost interest in showing hospitality to foreigners, we have all the more opportunity! We can welcome foreigners, show compassion, and so commend the gospel right here in America.
Some numbers will illustrate the scale of this opportunity. Since 1970 more than 35 million individuals have immigrated to the United States. And that’s not counting the more than 700,000 college students who come here to study each year, or the millions of illegal immigrants living in the shadows of our cities and towns.
You don’t need to live in New York or Los Angeles to reach out to newcomers. The largest community of Kurdish people in America is in Nashville, Tennessee. The biggest community of Somalis is in Minneapolis, Minnesota. Springfield, Virginia hosts the second largest community of Afghans in the Western Hemisphere.
And, of course, there are hundreds of thousands of college students from all over the world—a new flood every year. Even many smaller universities have substantial programs for international students. You might be surprised what nations God has brought to your doorstep.
AN EXAMPLE TO ENCOURAGE
The members of my own congregation, Capitol Hill Baptist Church, have embraced this evangelistic opportunity in our community. And I want to share some of what those faithful saints have been doing, not to say others should do exactly what we do, but to encourage similar kinds of faithfulness.
Our outreach to internationals started when one of our missionaries came home and spent a year living in Washington, D.C. During his time with us, the one thing we asked was that he help us survey the population of internationals living near our church. At the end of his time he concluded that students were the only significant resident population of internationals nearby. Most longer-term immigrants lived out in the distant suburbs. So our most significant ministry would be among students.
It so happened that there was a man in our church from Singapore who had himself been converted as an international student in the UK. One of our elders began to meet with him and asked him to think about how he might encourage more outreach from our church to international students. He began to host a Bible study in his home for international students. Later, English language classes were started in our church and on a nearby campus. More members got involved and began to meet one-to-one for practicing English by talking through the gospel. They were open and above-board in telling students this gospel conversation was our aim right from the start. As a result they had more opportunities to meet up with international students.
Eventually the effort grew until so many church members were involved that our elders decided to create the position of Deacon of International Outreach. This deacon would coordinate and give leadership to all the activity already happening: teaching English, hosting international students for meals, spending leisure time with them, picking them up from airports, and, most of all, meeting one-to-one to study through the Gospels with an interested student.
In the course of the following years we’ve had the joy of baptizing and adding to our church body several men and women who came to trust in Christ through these efforts. Others have believed and joined other churches in the area. Many have returned to their home countries where they are now witnesses for the gospel. Praise God!
ENCOURAGEMENTS FOR INTERNATIONAL EVANGELISM AT HOME
So here are a few observations drawn from our own these experiences.
1. Be willing to give pastoral leadership, but probably lightly.
First, be willing to give pastoral leadership, but probably lightly. As elders, we want to encourage the initiative of our members. We don’t want to press for top-down programs. But that doesn’t mean there’s not a role for our leadership, prayer, planning, and a few strategic decisions or conversations.
Certainly praying openly before your congregation that God might use your church to reach internationals is an obvious way to start. In addition, a few well-placed conversations with likely leaders in your church may stir up wonderful results. Growing in your own awareness of unrealized opportunities in your community cannot hurt either.
2. Let your international investments inform your local ones.
Second, let your international investments inform your local ones. We hope our church will find ways to reach out to Muslims, especially from nations where we have long-term missionaries at work. One of the best ways we’ve been able to do this is hosting our overseas workers for long-term stays when they are back in the States. And we want them spending months here, not days.
Having missionaries here with us who speak a people’s language and know their culture has been super helpful for building inroads among internationals. And if we are going to spend a lot of money sending people overseas to take the gospel to a particular people, we should certainly encourage our members to cross the street to reach the same people.
3. Be happy with unexpected fruit.
Third, be happy with unexpected fruit. As a church that is heavily invested in mission to the Muslim world, I wasn’t expecting that most of the fruit of our local work with internationals would be from secular East Asia. But as God would have it, that’s been the case. And that’s great! As our members have gotten to know students on local campuses, this has been the wonderful result and we’re delighted. Be strategic, but realize that the Spirit moves wherever he will.
4. Remember that all peoples need the gospel.
Finally, remember that all peoples need the gospel. Perhaps the best way to encourage a love for strangers is to keep reminding our people about the implications and imperatives that flow from the gospel. The only bridge we need to reach out to men and women from distant cultures is a reminder of our common state before God.
We all share the same parents. We all share in their sin. We all use our various cultures and man-made religions to hide from the true God and our guilt before him. We all need a savior from outside ourselves and from outside our culture. In short, we all need the gospel.
I know this may sound simplistic. But I’m convinced that, more than any program or effort or idea, the faithful preaching and careful application of God’s Word to the topic of cross-cultural evangelism is what God has most used to bring about the international fruit our own church is enjoying.
So pray for your own church. Lead them gently to embrace the nations around you. But build your church’s outreach all on a foundation of solid, gospel-informed love for every sheep who would come to hear Christ’s voice and follow him.
Andy Johnson is an associate pastor of Capitol Hill Baptist Church in Washington, DC.
Introducing: The Cross Conference [9Marks Blog: Building Healthy Churches] 2013-10-21 13:48
The Cross Conference is a new student missions conference coming to Louisville this Decemeber 27-30. It's headed up by Thabiti Anyabwile, Kevin DeYoung, John Piper, David Platt, Zane Pratt, David Sitton, and Mack Stiles, and there are dozens of breakout speakers from around the globe.
I'd encourage students, pastors, and anyone with a heart for missions to consider coming. The early bird registration rate for students ($100) ends 10/31.
To feel the heartbeat of the conference, check out this video narrated by Trip Lee:
A New Student Missions Conference from CROSS on Vimeo
Book Review: Manual of Church Order, by John L. Dagg [9Marks Blog: Building Healthy Churches] 2013-10-21 11:53
With a title like Manual of Church Order, the little book by Baptist John L. Dagg (1794-1884) has little hope of becoming an international bestseller. I usually associate the term “manual” with an irksome Saturday morning project (e.g., Electrical Wiring Manual). What’s more, “church order,” that is, ecclesiology, probably occupies a small, neglected corner of the minds of many church leaders. So neither “manual” nor “church order” sound all that exciting.
Yet Dagg’s 150-year-old Manual remains in print. This tells us something about the book’s clarity and importance.
Dagg recognizes that too many Christians ignore the sufficiency of Scripture in ecclesiology. For this reason, “We must return to the feet of our divine Master, and again receive his instructions. Let us, in the spirit of obedient disciples, inquire for the good old paths, that we may walk therein” (11). Those words from Jeremiah 6:16 ring true in our own day as well. We turn now to the basic outline and a few highlights.
Click here to continue reading.
How Can Churches Evangelize their Neighborhoods? [9Marks Blog: Building Healthy Churches] 2013-10-11 13:16

Once upon a time, churches were intensely local affairs. Christians attended the church nearest them, often walking or taking public transportation to the place where the congregation met. But North American communities and lifestyles have changed in the last fifty years, such that many people live, work, shop, play, and worship in different, and sometimes distant, places. Today, it’s rare to find a church where most of the members live in the neighborhood around the church building.
Yet churches still have a tremendous evangelistic opportunity in the people who live near the church building. After all, these neighbors walk and drive past the church building every day. They may wonder about what goes on when the church gathers. For non-Christians who don’t know any believers personally, the church down the street may be the biggest reminder of Christianity they see on a regular basis.
So how can a church be faithful in evangelizing the neighborhood when the members don’t live there? Some evangelical traditions have made a practice of “visitation,” knocking on doors in the neighborhood and trying to engage people in spiritual conversations. Sometimes this bears good gospel fruit, though cultural changes in recent decades have made this more difficult as many North Americans have become suspicious of strangers at the front door.
I serve my local church as deacon of community outreach, and our strategy for reaching the neighborhood around us is mainly one of long-term, patient faithfulness. Our goal is to build relationships with our neighbors that, over time, will make it easier for us to have spiritual conversations with them. These relationships also make our neighbors more willing to attend services and other events aimed specifically at engaging unbelievers with the gospel.
The basic principle behind this strategy is simple, and it’s one that any church can follow: engage your neighbors by taking an interest in what they care about. Building common ground is easy when you participate side-by-side in community organizations, service projects, family events, block parties, yard sales, and the like. Common interests are one of the most powerful tools for building friendships that can enable spiritual conversations to take place.
My church is located in a historic urban neighborhood that has a well-defined identity, and many of our neighbors have common interests. Neighborhood associations are popular and prominent in the life of the community, and events like street fairs, art shows, music festivals, park cleanups, and community yard sales are common. We engage our neighbors by having church members volunteer for these events, host booths, and attend neighborhood association meetings. We also invite the community to a couple of evangelistic events at Christmas: a service of lessons and carols with a brief evangelistic sermon, and a sing-along production of Handel’s Messiah.
But even if your church doesn’t have the advantages that my church’s neighborhood affords, you can still engage your neighbors by showing you care about what’s important to them. Just spend some time thinking about what your neighbors value, what they spend their time and resources on, and think about ways you can build relationships with them through those things.
If your church is in a lower-income area, your neighbors’ biggest concerns are likely to be some of their most basic needs: food, shelter, jobs, transportation, education. Your members might help meet some of these needs, and thereby gain neighbors’ trust and attention, through soup kitchens, clothes closets, literacy programs, and such. Check out the July-August 2012 9Marks Journal for articles by Mike McKinley and others on using mercy ministries as vehicles for evangelism.
My father pastors a church in Ohio in a middle-class suburb with a lot of families, and many of these neighbors’ lives revolve around their kids. So the church hosts some events throughout the year that provide activities for the kids and expose neighbors to the gospel. The church puts on a Vacation Bible School every summer that is advertised to the neighborhood. They host a big Easter egg hunt for the kids of the neighborhood, and someone tells the resurrection story with a clear gospel presentation for the whole crowd.
Evangelizing the neighborhood where your church meets takes a degree of strategy and effort, especially when your church’s members don’t live there. But it doesn’t have to be difficult, and you don’t necessarily need a huge budget. Just find ways to build relationships with your neighbors by showing your members care about some of the same things they care about.
Make sure your members understand that, while it’s always good to love our neighbors and build relationships with them for a number of reasons, we love them best by sharing the gospel with them. When good gospel conversations do happen, engage the whole church in praying that they would bear fruit and that the Lord would use them to save your neighbors.
Jeff Cavanaugh lives with his wife, Andrea in Louisville, KY, where he is a member and deacon of Third Avenue Baptist Church. He has a Master of Divinity from The Southern Baptist Theological Seminary and works full-time in Louisville.
Reaching the “Converted” [9Marks Blog: Building Healthy Churches] 2013-10-10 11:29

Some of our most obvious evangelistic opportunities are with the people who are members of our churches. You already have a relationship with them. You already have the advantage of consistently telling them the gospel. You also have some God-ordained opportunities to personally point them to Christ.
Paul warned the elders of the church at Ephesus that fierce wolves would come in among them and seek to do great damage to the flock (Acts 20:29). Christ warned several of the churches in Revelation 2-3 that they had unbelievers in their number. If these churches had unbelievers in them, we probably have some in ours too. But, how do we reach them?
HOW TO REACH UNCONVERTED MEMBERS
I am assuming that you are faithfully preaching the gospel and pointing your people to Christ. The effect of faithful gospel preaching is like napalm: it has a way of wiping out everything else. But, in order to conquer, you still need to ground troops. So, while you are joyfully preaching Christ, pursue these steps as well.
1. Pray about the conversions of your church members.
First, pray about the conversions of your church members. Pray that God would distinguish the posers from the possessors. Most of you, I would assume, publicly pray at the beginning and conclusion of your preaching. These are wonderful opportunities to pray about this critical matter—that people would not rely on their membership as giving them a right status before God, but that all would be truly repentant and trusting in Christ.
2. Preach about the conversion of your church members.
Second, preach about the conversion of your church members. If you are preaching expositionally, you can’t preach too many sermons before you run into the issue of false conversions. In your preaching, illustrate the point with stories from your own church family.
When someone gets baptized, we give them the opportunity to explain the gospel and how they came to faith in Christ. Last month, David told our church family how he had pretended for years to be a believer. His story is a great example that I refer to often.
3. Be aware of this in counseling.
Third, be aware of this in counseling. Devin (not his real name) and his wife met with me for some marriage counseling. Devin was not all that interested since, as he eventually revealed, he thought he had found someone else. One Sunday, I stopped him after the service and told him that if continued down that road, he needed to know that he could no longer confidently claim to be a follower of Christ. In fact, his determination to pursue this adulterous relationship may be an indication that he had never become a genuine follower of Christ.
Devin did not repent, but Greg (not his real name) did. Greg met a girl on a business trip and was ready to leave his wife and kids over her. I sat at his kitchen table one night and asked him what would it be, Christ or the girl, because he could not have both. Although Greg had professed faith and joined the church many years before, his life had demonstrated very little gospel fruit. Greg bowed the knee of his heart to Christ and by the grace of God, he was not only redeemed, but his marriage was rescued.
4. Be aware of this in hospital visits and other life and death situations.
Fourth, be aware of this in hospital visits and other life and death situations. Chuck (his real name) was in the hospital. The doctor had just told him that there was nothing left that could be done for his heart. He had already outlived the expectations, but the end was near. Chuck was a successful businessman and had been involved in many Christian organizations. In previous churches he had served on boards and taught classes. Now he was dying and he was terrified.
Chuck carried around a secret that very few people knew. During World War II he flew bombing missions over Japan, dropping thousands of pounds upon that country. He knew that he had killed hundreds if not thousands of people. On his 24th mission, his plane was shot up pretty badly, but he was able to get it back to base. His co-pilot, however, died. Chuck was eligible to go home after his 25th mission, but he was so angry about the death of his co-pilot that he signed up for another 25 missions and then yet another 25 missions so that he could kill more Japanese. And he did. After 76 missions, he finally went back home.
On his way back to Michigan, he was at a base in California where he met some Japanese prisoners of war. Some of them were very kind and told him that they did not want the war. They just wanted to go back to their home as well. They showed him pictures of wives and children. Chuck’s anger turned to fear. He assumed that he had killed some of their wives and children. He began to realize that he had not only killed civilians, but he had signed up to do it.
Now, sixty years later, the reality of facing God revealed his deepest fear. He would die and be condemned to hell. Chuck finished his story, tucked his knees under his arms, turned away from me and stared at the wall. His frail body made even a hospital bed look big. Chuck had heard me preach the gospel for years. But that day it was obvious that while he thought it was true, it just wasn’t true for him. His case was different.
I sat silent and tried to imagine the weight of his guilt and then said, “Chuck, you are a big sinner, but Jesus is a bigger Savior than you are a sinner.” Chuck responded like he had been hit by lightning. He looked at me like he had heard this for the very first time. His eyes got big, his face was animated, and he said, “That’s it, isn’t it?! Jesus is a bigger Savior than I am a sinner.”
Chuck died two weeks later. The joy of his life in those last two weeks made it evident to everyone who visited him that his chains were broken. His heart was free.
Your members will let you in to some of their most private thoughts. You may discover that what they need is to believe in Christ—for the very first time.
Bob Johnson is the senior pastor of Cornerstone Baptist Church in Roseville, Michigan.
Three Lessons for Cross-Cultural Evangelism [9Marks Blog: Building Healthy Churches] 2013-10-09 11:26

In our church in Dubai, we have been amazed to witness conversions of people from Eritrea and Uzbekistan, Syria and South Africa, Scotland and Spain, Iran and India, the Netherlands and Bolivia, Germany and China, and more. They are from religious and non-religious backgrounds, traditional and progressive, Muslim and Hindu, young and old.
What is the key to unlocking the hearts of these people from such an array of cultural and religious backgrounds?
The answer is, there is no “cross-cultural key.” In our evangelism, we don’t do anything differently here than we would anywhere else. Our evangelistic methods are singularly uncreative. To suggest that some people are easier to convert than others is foreign to the Scriptures. All of us, by nature, are “far off.” And so in our evangelism we must bear witness and pray and await the sovereign move of the Spirit.
There is no “key” into a spiritual morgue.
But this doesn’t mean that cultural diversity is irrelevant to evangelism. Most of the world’s cities are becoming more and more ethnically diverse. With 202 nationalities in its labor market, Dubai is ahead of the curve in this area. The world has descended on Arabia, bringing with it both challenges and opportunities for evangelism.
THREE LESSONS
Here are three lessons we have learned living and ministering in an ultra-multi-cultural environment:
1. Communicate clearly.
First, communicate clearly. Muslims are taught from childhood that God has no Son. Hindus deny there is one transcendent Creator who grounds all existence and morality. Secular humanists think religious truth is relative. So, whomever we’re speaking to, we must define our terms clearly. With Muslims, we unpack what the Bible means about God’s Son: not that the Father and Mary physically produced offspring akin to Zeus and Danae, but that the eternal image of the invisible God, who preexisted the universe, came down himself and took on flesh.
With Hindus, we work to explain a moral universe, one where good and bad are defined by God’s character and his revealed will. There’s no use talking about “sin” (Rom. 3:23) or pointing people to the “Son” (John 3:16), unless and until we have unpacked these freighted concepts. In multi-cultural settings we must, as D. A. Carson has said, “start farther back in our evangelism to provide more of the Bible’s story line for the good news to cohere…so we have to unpack more of the doctrine of God, and thus of the Son, to a generation that knows nothing of the Trinity.”[1]
This is why, when Thabiti Anyabwile publicly dialogued with Muslim imam Shabir Ally in Dubai last spring, his opening statement was a 20-minute survey of Old Testament theology leading up to the life and ministry of Jesus. Unless the listeners grasped the storyline of the Bible, the significance of the atonement would be lost on them.
This is simply clear communication, which is all the more important when we live among people who are biblically illiterate and inoculated against a biblical worldview.
2. Proclaim the Word.
Second, proclaim the Word. James teaches that God “brought us forth by the word of truth” (James 1:18). Wherever we are, the agent of regeneration is biblical revelation, read and proclaimed. This is why, in our evangelism, if the person can read, our goal should be to study the Bible with them, regardless of their culture.
“Friendship evangelism” is increasingly popular in the Middle East and many other places, because of the (mistaken) impression that we cannot or should not directly and clearly communicate what the Christian message is, but rather we should allude and insinuate until the friend shows an openness to hearing more. Friendship evangelism emphasizes that we must earn the right to speak the gospel to another person. Of course, we ought not use people merely as evangelistic projects. But, as one evangelist here told me, there is a danger of too much friendship and too little evangelism. Excessive concern about context and techniques will tend to overshadow the command simply to “preach the Word.”
3. Use the local church.
Third, use the local church. Whatever continent you’re on, the church is a gathering of people who are indwelt by God’s Spirit, and who gather weekly for preaching, singing, prayer, and the ordinances. Paul expected the weekly assembly not only to build up the believers, but also to convict non-believers who attended (1 Cor. 14:25).
Over the years, several people from “restricted access” or “closed” countries have quietly attended our church, or even walked into our building during the week and asked to learn about Jesus. Or they have called the church office, identified their religion, and asked to meet with someone to consider the claims of Christ. We were all too happy to oblige—not to pressure anyone, but to offer them friendship, true and clear explanations of the gospel, and the opportunity to observe the three-dimensional display of the gospel that is a local church.
In many of these cases, these people were born again and joined together with us. They not only heard and understood the gospel, they saw how the power of Christ changes individuals and influences entire communities that have little in common except Christ. The church, then, is the confirming echo of the gospel that is being proclaimed.
FOREIGN TO ALL CULTURES
Increasingly, global cities are home to multi-national churches that worship in English, the lingua franca of our day. These churches reach into countless national and ethnic groups, even through English as a second language. When expatriates return to their ancestral homes, they take the gospel back with them.
It’s true that multiculturalism poses challenges for evangelism. However, regardless of where we’re from, we must remember that the gospel is foreign to all of our cultures. For all our diversity, we are still sons and daughters of Adam and Eve, in need of the one remedy that only Jesus could secure: redemption, the forgiveness of sins.
Churches in multi-ethnic settings must work hard to communicate clearly, with due regard to careful biblical theology. We must be centered on scriptural truth that will slice through all manner of cultural and religious barriers. And we must hold up the church as the display of the gospel to the nations.
John Folmar is the senior pastor of United Christian Church of Dubai.
[1] Jesus the Son of God: A Christological Title Often Overlooked, Sometimes Misunderstood, and Currently Disputed (Crossway, 2012), p. 85.
Evangelism across Economic Boundaries [9Marks Blog: Building Healthy Churches] 2013-10-08 12:00

A FEW LESSONS
Here are a few things that I’ve learned from leading a church that is trying to reach out to folks from different backgrounds.
1. We’re not all that different
First, we’re not all that different. It can be intimidating to try and build relationships with people who experience life differently, especially in things that can seem so important: clothing, work, education, expectations, living arrangements. But in reality, such matters are a tiny fraction of what makes us who we are.
You probably have a tremendous amount in common even with people that seem very different from you. Everyone—perhaps with the exception of a few Brits I’ve known—wants to be loved, known, and accepted. We all love our children and are grateful to people who are kind to them. We are all prone to worry about what the future holds. But most importantly, we are all “in Adam” and in desperate need of a savior (1 Cor. 15:22).
Churches who want to reach out across socio-economic boundaries need to make their first step towards others on the basis of these commonalities. It’s fairly simple: treat other people with unfeigned sympathy and respect, as fellow travelers to the grave (to steal a phrase from Dickens). This approach will help prevent the sense of condescension that flavors and spoils a lot of well-meaning attempts to reach across class lines.
2. It helps to be a blessing
Second, it helps to be a blessing.
You really don’t want to build your outreach solely on the basis of giving people things—food, money, gas cards. Those things can be helpful, but if that’s all you do, you are giving people the chance to come for just the handout and remain unchallenged by the source of the love behind the handout. That doesn’t mean that you can’t use the resources the Lord has given you to help build connections with others. A few examples:
In all of those cases, we were able to leverage resources that we had to bless people, connect with them, and eventually share the gospel.
3. Environment matters
Third, environment matters. If you want to reach out to people who are less affluent and privileged than you are, look around at your church and your life. Try to imagine how someone less fortunate than you (sorry, I’m running out of euphemisms) might perceive them.
Do your sermon illustrations assume that everyone has been to college? Or owns a car? Or has access to a computer or cable TV or designer clothing? These kinds of things speak volumes to people about whether or not they are truly welcome to be part of your church.
Is your house—its size, neighborhood, furnishings—intimidating to someone with few resources? Would it immediately make them feel uncomfortable or shabby? If so, you will probably have to work through extra layers of defensiveness in order to reach people.
Is your home in a location where poorer people (who may not have a car) can walk or take public transportation? If not, it will be more difficult to be hospitable.
4. Know who you are talking to when explaining the gospel
Finally, if you want to reach out to people with different backgrounds, consider how you are explaining the gospel. To be clear, the message must remain unaltered. All men, women, and children need to hear of their sin, God’s holiness, the death and resurrection of Christ, and the need for repentance and faith. But you may need to find new methods of delivering that message for people who are not comfortable with the English language or with reading as a way of gaining information.
If I am sharing the gospel with an educated professional from northern Virginia, I may well invite him to read a book with me in order to help him investigate the claims of Christ. And certainly, there are some poorer folks who are well educated and enjoy reading. But we also need to have other ways of communicating for people who are not readers. Two examples: using videos (like Christianity Explored) or stories (I like the ones being used at Soma Church in Washington) to communicate the movements and themes of Scriptures.
Mike McKinley is the senior pastor of Sterling Park Baptist Church in Sterling, Virginia, and is the author, most recently, of Did the Devil Make Me Do It? (The Good Book Company, 2013).
Evangelism in the Workplace [9Marks Blog: Building Healthy Churches] 2013-10-04 13:19
As cultural opposition toward Christianity grows, what is its effect on your evangelism at work? Are you more faithful or more fearful?
You could hardly be blamed for being more fearful. The rapid advance of social liberalism and human resources policies promoting workplace “tolerance” only exacerbate the two fears we commonly cite for not sharing the gospel with our co-workers: fear of social harm and fear of career repercussions, like job loss or career stalls.
Evangelism has always been hard. If there is anything new about our challenges today, it’s how emboldened the opposition seems to be. Non-Christians used to say “To each his own.” Now they are just as likely to accuse us of stupidity (“Seriously, you don’t believe in evolution?”) or hateful bigotry (“How dare you say homosexuality is a sin?”). Employers increasingly do rigorous social media background checks before making hiring and promotion decisions. How long before companies who are fearful of workplace harassment and discrimination pass over the more visible Christian for someone who makes fewer waves?
In spite of all this, I am so grateful for the brothers who feared God more than man and shared the gospel with me. My own faith is the fruit of workplace evangelism.
LOST, AND FOUND IN THE WORKPLACE
Twelve years ago, I was a researcher at a mid-sized consulting firm in Washington, DC. I was a self-confident, self-sufficient, professionally-prospering Hindu. You wouldn’t have assumed I was spiritually uncertain. Frankly, I didn’t know I was spiritually uncertain. What I was not was a guy who was actively seeking Christ.
Enter my Christian colleague Hunter. Well-known and well-liked around the office, Hunter was a high-performing sales guy with a range of interests. Someone told me, “He’s a Christian, ya’ know.” Neither one of us knew for sure what that meant, but both of us believed it was relevant enough to add a knowing, “Huh.”
I did know Hunter didn’t fit the mold of a Christian that I had mentally constructed. Christians were nice, old-fashioned, hypocritical, one-note tunes. Hunter wasn’t that. So I started watching him.
We became friends. We spent time together and talked about a range of topics—The Simpsons, Lord of the Rings, Christ, Krishna, coffee, work. While the Lord used Hunter to pursue me, I never felt like a project, just a friend. As only God can do, he providentially arranged for Hunter to be there at the same time that God orchestrated a spiritual crisis in my life. And he gave Hunter the wisdom and boldness to speak truth into my life when I needed it most.
BEHAVIORS OF A WORKPLACE EVANGELIST
While young in the faith himself at the time, there is much about Hunter’s example that any believer can apply in a workplace setting.
1. Put Christ on the Table
First, put Christ on the table. Because it can be rare to meet Christians in the workplace, it is essential that people in your office know that you are a follower of Christ. That way you can make yourself available to weaker believers and an example to non-believers. It was a non-Christian colleague who told me about Hunter’s faith. Obviously we should not do this obnoxiously or irresponsibly, but by recounting your weekend, describing a Bible study that you are in, or sharing how you pray for others, people will soon know.
2. Work with Excellence
Second, work with excellence. When you put Christ on the table, expect to be studied by your peers as I studied Hunter. Work in a way that reflects the creativity, purpose, and goodness of God. Demonstrate faithfulness and integrity. Work “without grumbling or complaining” (Phil. 2:14). Submit to those in authority, and serve humbly.
This in itself isn’t evangelism, but the content of our lives at work should reinforce, not undermine, the content of the gospel message we share.
3. Love your Peers
Third, love your peers. Invest in friendships with non-Christians in your workplace, not perfunctorily as “projects,” but lovingly as those made in God’s image. Don’t underestimate the importance of trust. Consider that it was a year and a half after Hunter and I met that we studied the Bible together and God gave me ears for the gospel.
Use your lunch break strategically. As you’re able, make generous use of hospitality, where you can share your life with a colleague away from the office and the usual chit-chat and office banter.
4. Prepare to Evangelize
Fourth, prepare to evangelize. As silly as this may sound, be sure you know how to easily explain the gospel. Practice if you need to.
When the Lord provides an opportunity, you don’t want your inner voice screaming at you for being unclear—you want your mind free to listen to your colleague and what they are struggling to understand. After all, it is the gospel that saves, not our quick wit and strong grasp of apologetics. I praise God for Hunter’s clarity, boldness, and trust in power of the gospel.
5. Pray
Fifth, pray. Pray for your colleagues regularly. Pray for good opportunities to share the gospel. Pray that you would grow in boldness. Pray that God would be big and man would be small—we’re all guilty of getting the two mixed up.
And invite brothers and sisters in your church to pray as well. Hunter later told me that his men’s Bible study group was praying for me from the moment I asked him about his Christian faith.
A CALL TO FAITHFULNESS
As workplaces grow more hostile to Christianity, these basic practices will be all the more essential. The Lord has been kind to answer my many prayers for good opportunities and the words to speak. Being known as a Christian, living out my faith professionally and interpersonally, and loving my colleagues more as God’s image-bearers has gained me opportunities to speak openly of my faith. And, in his amazing grace, God has chosen to use me to bring a colleague to faith.
We should expect the Lord to answer our prayers and grant us opportunities to speak of Christ, so pray for boldness. And be willing to spend your relational capital. God has put you where you are for a purpose.
Ashok Nachnani is an elder at First Baptist Church in Durham, NC, and a strategy executive at a multinational energy management company.
Coming Soon: 9Marks at Cedarville [9Marks Blog: Building Healthy Churches] 2013-10-02 12:14
If you're within striking distance of Cedarville, Ohio, don't miss the upcoming 9Marks at Cedarville conference!
Where: Cedarville University in Cedarville, Ohio
When: October 29-30, 2013
Topic: A biblical understanding of the gospel
Speakers:
More Information: click here.
Ten Steps to a More Fruitful Sabbatical [9Marks Blog: Building Healthy Churches] 2013-09-20 11:08
I recently returned from sabbatical. My church totally relieved me of duty for the months of June and July. I was banned from Sunday services at our church and was kept in the dark about pastoral issues they faced during this two-month period.
Leading up to this time, I sought counsel from many pastors who had been given similar time off. I was struck by how many shared of different regrets once their time was done. So I tried to use my Sabbatical in the most fruitful way possible. Here are a few lessons I learned.
1. Delight in your wife. Have plenty of date nights. Care for her. Study her. Learn from her. Laugh with her. Enjoy her. Reflect on your years of ministry together. Realize she needs this time as much as you do. Resolve to make it a great benefit to her soul. Seize time to delight in her while the busyness that often cuts into your time together is temporarily relieved.
2. Enjoy your kids. I have never before had such an extended period where I can focus on time with my kids. I needed to make sure they were not only a primary focus, but that my heart was taking in this time with them and truly enjoying them. Many pastors expressed regret to me on this front. So we spent time at the pool, parks, out of town a bit, reading, wrestling, laughing, riding bikes, and whatever else they wanted to do.
3. Be intentional with spiritual disciplines. I committed to have times of reading God’s Word that were long and covered large portions of text. I usually spend most of my time “staring at the trees” for sermon preparation; for this break I allowed “the forest” to feed my soul.
I also had intentional times of silence and prayer for the sake of my own soul, asking God for guidance on a vision for our church for the next ten years, as I’ve just finished up my first ten as pastor. Additionally, I renewed a helpful discipline I’ve neglected: journaling. Embrace the basic spiritual disciplines we exhort our people to engage in that we can often let slide in our own lives.
4. Be consistent with physical disciplines. Commit to sleeping eight hours a night. Try to renew regular exercise—for me, this meant a three to four day a week workout plan. And resolve to eat well. If you do none of these well in your normal grind, a sabbatical can be a great time to recommit to stewarding your body and energy well. I lost ten pounds on my sabbatical and was reminded how much sleep I actually need to be at my best to serve the Lord. Do not underestimate how poorly you care for your body during the grind of ministry.
5. Be mentored by a faithful dead pastor. Dead pastors from different moments in history can teach us about pastoral ministry in ways modern pastors cannot. I chose the great 18th century English Particular Baptist Andrew Fuller (1754-1815) to mentor me during this time via his writings. It was so encouraging! Pick one, then immerse yourself in his life and ministry and allow him to teach you.
6. Learn about preaching from a faithful living pastor. I chose Ted Donnelly, who pastored in Northern Ireland for over 35 years until his health recently declined. He is known in Britain as one of the most gifted, Spirit-filled preachers in the last half century. I listened to his sermons and learned much. God also fed my soul through his Word in the process. Choose someone you don’t know very well but would be a helpful instructor to push you to grow in your preaching.
7. Visit other churches. It can certainly be restful and encouraging to worship among your people with your regular pastoral duties relieved. But the inevitable conversations that will arise can make a Sabbatical less of a break if you spend Sundays in your own church. So I made sure my responsibilities at church were covered so that I could worship at other churches for the entire sabbatical.
If you go to other solid churches where the Word is preached you will experience Christian fellowship. There is much to learn from other churches and pastors. You may experience something in their public gathering you then choose to bring back to your church. If you do not have many choices, pick a couple of solid churches during your sabbatical where you can simply attend, relax, and be fed while sitting with your family.
8. Put off the tasks you normally put on. A Sabbatical will not be truly restful if you hang on to what normally wears you down. This is why my fellow pastors banned me from writing a book or preaching anywhere, both of which are a normal part of my ministry. Although many take sabbatical time to write—which is fine for some—my fellow pastors were right to forbid me from doing so. Make sure you are honest with yourself about the things that wear on you. And make sure set them down for this time, even if they are things you love to do.
9. Play golf. Golf is relaxing yet humbling for most of us. There are layers of reasons this is good for your soul. I shot some of my best rounds of golf in years during my Sabbatical and beat my very competitive father for the first time in my life. Clearly, the favor of the Lord was upon me. If not golf, find some other relaxing, humbling way to have fun that’s tough to fit into your regular grind.
10. Truly rest. I typically don’t rest well. But I realized through others’ counsel that if I came to the end of my time off and my wife and I did not feel refreshed and rested, we would have defeated the purpose of this gift from our church and squandered this opportunity. Whatever will help you rest from the rat race of your regular labors and refresh your soul is what you should do.
If you are planning for an upcoming sabbatical, I hope this begins a helpful conversation between you and your fellow pastors about what would be the best way for you to benefit from this gift. Be intentional. Involve others in your church to help determine the best way for you to spend your time. Listen to your wife’s input. And pray God would grant you to rest well and wisely, so that fond memories vastly outweigh regrets when you return to the normal routine of ministry.
Brian Croft is the senior pastor of Auburndale Baptist Church in Louisville, Kentucky and the founder of Practical Shepherding. Along with his wife Cara, he is the author of The Pastor’s Family: Shepherding Your Family through the Challenges of Pastoral Ministry (Zondervan, 2013).
Book Review: Blessed: A History of the American Prosperity Gospel [9Marks Blog: Building Healthy Churches] 2013-09-10 09:02
Flip to just about any TV channel with religious programming and you are likely to encounter the prosperity gospel. From Joel Osteen’s perma-smile to T. D. Jakes’s mopped brow, from Jan Crouch’s bouffant cotton-candy hairdo to Benny Hinn’s Nehru jackets, America’s electronic preachers tell us there is a God of inexhaustible abundance ready to bless us with our own personal miracle. Whether our troubles are financial, physical, or emotional, God will change our fortunes if we will just pray in faith for the desired outcome, proclaim it ours, and then act on the certainty of its arrival in our lives.
Of course, it helps to sow seeds if you want to reap a harvest, so a gift given to the ministry is a tangible sign of our faith that God will do as we say. And the bigger the gift, the larger the faith. Lest we doubt this simple spiritual formula, we need only look at the extravagant lifestyles with which the good Lord has blessed prosperity preachers. It’s all so straightforward and appealing, as American as mama’s apple pie and a 30-year mortgage. Evidently, we can’t get enough of the stuff.
THE ORIGINS AND DEVELOPMENT OF THE PROSPERITY GOSPEL
In a thoughtful and engaging work, Kate Bowler unravels the origins and development of the prosperity gospel into a multi-billion dollar industry. Although there are several varieties of prosperity gospels with subtly different animating convictions and practices, Bowler sensibly lumps them together as birds of a feather, a range of species in the same genus. “Word of Faith,” “Positive Confession,” “Health and Wealth,” and so forth, they all share a bedrock conviction that God chooses to bless his children with material prosperity in body, mind, and brokerage account, awaiting only our willingness to get on board.
Click here to continue reading.
Pastors’ Forum: Four Approaches to Evangelism Training [9Marks Blog: Building Healthy Churches] 2013-09-09 10:32

We asked four churches to tell us how they equip their people to evangelize. Here are their responses. Answers from University Reformed Church, Sterling Park Baptist Church, the Village Church, and the Church at Brook Hills.
Ben Falconer, University Reformed Church, East Lansing, Michigan
If we’re going to evangelize faithfully, we need to talk, pray, and be challenged about it. With that in mind, at University Reformed Church we attempt to keep evangelism at the forefront of our ministry as much as possible.
The foundation is laid with regular admonition and encouragement from the preached Word on Sundays. As often as the text gives us opportunity to trumpet our responsibility to be heralds of the good news, we take it. Evangelism and praying for the lost are repeated applications that we as pastors make from the text.
Another way we teach on evangelism is by including it in our new members class. We want those interested in the church to hear right from the beginning that the Scriptures expect believers to share their faith. We take class time to walk through a gospel tract that our senior pastor Kevin DeYoung and the staff developed a number of years ago. Then we give each new member time to practice sharing with a partner.
A third way we have sought to equip the congregation in evangelism is by making it our theme for a given year. We have identified four basic disciplines of the Christian faith (prayer, Bible study, missions, and personal evangelism) and we aim to focus particularly on one each year. For each theme, we offer specific training, have a corresponding sermon series, and provide other opportunities for practice or accountability. When we focused on evangelism a few years back, we also had the entire church read through Mark Dever’s book The Gospel and Personal Evangelism and discussed it in our small groups.
Ben Falconer is associate pastor at University Reformed Church in East Lansing, Michigan.
Mike McKinley, Sterling Park Baptist Church, Sterling, Virginia
At Sterling Park Baptist Church we offer training to our people on how to share the gospel with the hurting and needy. Our mercy ministry and outreach to “at-risk” youth generate a lot of gospel opportunities, but we realized pretty quickly that most of our members weren’t naturally comfortable interacting with and sharing Christ with people who seemed so different.
We try to train our people to listen and ask good questions so that they can identify how this person understands what has gone wrong in their life and what they think will fix it—that is, their version of the Fall and Redemption. Once our member understands how that person understands their “story,” they can share the true story of Christ with them: their real problem is that they are enemies of God, but the good news is that God has made a marvelous salvation available through Christ.
We also have about 30 minutes set aside in our Sunday evening service to pray for evangelistic opportunities that have come up in the previous week, or that we hope will come up in the following week. Members share about conversations that they’ve had or plans they’ve made to share Christ with people in their lives, and then we ask God to give more opportunities to us and bear more fruit through us. This helps make evangelism seem like a normal part of the Christian life, rather than something done by the professionals. It also drives home the point that evangelism begins with prayer.
Mike McKinley is the senior pastor of Sterling Park Baptist Church in Sterling, Virginia, and is the author, most recently, of The Devil Made Me Do It (Good Book Company, 2013).
Josh Patterson, The Village Church, Flower Mound, Texas
At The Village Church, we try to equip our people to fulfill the Great Commission in three ways: we model it, preach and teach it, and celebrate it.
First, the church leaders model evangelism. We are not asking our people to be involved in something that we ourselves are not doing. The pastors and elders are sharing Christ with their neighbors, friends, and family members.
Second, the pastors preach it and teach it. The preaching of the Word stands as a constant reminder of God’s call for his church to be his ambassadors in the world as he makes his appeal through us. Also, we teach evangelism in a variety of contexts. A primary equipping venue for us is our home group groups. Here we have a “multiplication guide” that walks a home group through six-month evangelism training course.
Finally, we celebrate it. What is celebrated is cultivated. And a culture of evangelism is stronger than any evangelistic program. We celebrate evangelism through stories of conversion and faithful members who bear witness to Christ. Four times a year we have “Celebration Weekends” where the bulk of the worship gathering centers around the proclamation of the gospel through baptism. At The Village, we ask those who were integral in the conversion of the individual being baptized to perform the baptism. In other words, our members baptize those they lead to Christ.
Our desire to celebrate, teach and preach, and model evangelism serves to reinforce this biblical call that for disciples of Jesus, evangelism should be normal.
Josh Patterson serves as Lead Pastor for Ministry Leadership at the Village Church in Flower Mound, Texas.
J.D. Payne, The Church at Brook Hills, Birmingham, Alabama
At Brook Hills, we recognize that the best evangelism equipping strategy is multifaceted. This requires:
While we spend a great deal of time in multiple venues talking about sharing the gospel, we know it is not enough to just talk about evangelism. All of our elders are required to develop and submit an annual personal disciple-making strategy, and all of our members are encouraged to do the same. This past year, two of our pastors preached a several-week series on personal evangelism. At least twice a year, we offer a six-week personal evangelism training, with plans to offer it three times per year starting in 2014. I also do a weekly 5-10 minute vodcast called “Multiplication Matters,” addressing issues related to evangelism.
J. D. Payne is Pastor of Church Multiplication at The Church at Brook Hills, Birmingham, Alabama
Why Christians Should Wax Weepy with Wax [9Marks Blog: Building Healthy Churches] 2013-09-05 15:38
I am grateful for Trevin Wax’s blog post “I Weep for Miley.” And I want to tell you why: it joins a genre of Christian literature that is rare but appropriate—the cultural lamentation.
There is plenty of Christian writing (and teaching) in which the writer (or teacher) laments his or her own failings. And there is lots of cultural criticism to be found on any given day in the Christian precincts of the twitterverse or blogosphere. All this can be done well or poorly.
But what makes the lamentation distinct as a form of criticism is that it more obviously exposes the writer's heart posture to be one of love. The writer hurts, mourns, even weeps because a person or a people whom the writer loves are destroying themselves with folly and sin. Beholding such sin, the writer cannot help but say, “Oh, please, no, don’t! God, help! Friends, why would you…”
America is not the new Israel, nor is any other nation to be equated with the people of God. But I think there is a place for the Christian citizens of a nation to behold the self-destructive forces of sin at work in their nation and then to weep, just as one might weep for a non-Christian parent. Our nations—at least the better ones—have reared, protected, and provided for us in our growth into adulthood (see Acts 17:26-27). We are rightly affectionate for them.
These are the people who came to our birthday party at age 7, and played on our soccer team at age 11, and showed up with us for freshman orientation at college, and sat in the cubicle next to us in the office. They have built our streets and watched our borders. These are our friends and guardians.
So there should be something inside of every Christian that mourns for these friends when we see them hurting themselves by rejecting Christ and believing the lies that lead to death.
“Ah, a people loaded with guilt,” the Old Testament prophets would say.
And David: “I weep because your laws are not kept.”
Jesus, too: “Oh, Jerusalem, Jerusalem, how I long to gather you as a mother gathers her chicks.”
Yes, the unteachable and the self-righteous will always reject our lamentations because a lamentation involves moral evaluation, and moral evaluation defiles the temple of the sacred self.
Yet everyone laments something. Even that most holy priest of the sacred self, Walt Whitman, offers a wonderful specimen of the genre in his poem, “I Sit and Look Out”:
I sit and look out upon all the sorrows of the world, and upon all oppression and shame;
I hear secret convulsive sobs from young men, at anguish with themselves, remorseful after deeds done;
I see, in low life, the mother misused by her children, dying, neglected, gaunt, desperate;
I see the wife misused by her husband—I see the treacherous seducer of young women;
I mark the ranklings of jealousy and unrequited love, attempted to be hid—I see these sights on the earth;
I see the workings of battle, pestilence, tyranny—I see martyrs and prisoners;
I observe a famine at sea—I observe the sailors casting lots who shall be kill’d, to preserve the lives of the rest;
I observe the slights and degradations cast by arrogant persons upon laborers, the poor, and upon negroes, and the like;
All these—All the meanness and agony without end, I sitting, look out upon,
See, hear, and am silent.
In certain respects, Whitman’s conclusion is appropriate. Meanness and agony should leave us dumbfounded. In other respects, the Christian knows that God alone is the one before whom we are utterly silent, and that we do have something to say in the face of evils committed or endured:
Oh, my fellow citizens, look to Christ!
Look to him for healing from your woe,
and for pardon from your guilt!
How my heart aches because you will not look but choose death!
New Interview with Russell Moore on Religious Liberty and Ethics [9Marks Blog: Building Healthy Churches] 2013-09-05 14:16
We just released a new interview with Russell Moore on religious liberty and ethics. In it, Dever and Moore discusses Moore's new role, rap, relevance, and why "it's even worse than it appears, but it's alright."
Yes, that is a Grateful Dead quotation. A 9Marks first, if I'm not mistaken. What more reason do you need to listen?
Evangelism Tool Review: The New World Gospel Presentation [9Marks Blog: Building Healthy Churches] 2013-09-05 00:01

InterVarsity’s “New World Gospel Presentation” is an evangelism outline “designed to lead others to make a decision for Jesus Christ and join his mission to heal the world.”[1] As of today there are training materials on their website, Vimeo and YouTube. Additionally, they have developed a free app (iOS and Android) to help illustrate the main points of the presentation during a gospel conversation.
ENGAGING THE “FOUR WORLDS”
The presentation is built on the premise that most people ache for a better world. The outline works through the paradigm of a world, using four worlds to communicate the biblical story. These four worlds are the four points for the conversation. Like many other presentations, they aim to frame redemptive history in their main points:
World 1: The world and all that’s in it was designed for good.
World 2: We—and the world—were damaged by evil.
World 3: Jesus came to restore the world and everything in it to what God intended.
World 4: Jesus invites us to join him and his community to heal the world.
InterVarsity ambitiously attempts to pull off the evangelistic version of a hat-trick with this presentation. They aim to listen to people’s stories (especially their scars and wounds), frame them within the context of the Bible’s story, and clearly communicate how Jesus answers their aches and pains. All of this they do while aiming to be faithful, winsome, compelling, clear, and understandable. Do they pull it off?
In communicating the first two worlds they do a fairly good job showing the divine design for creation and the problem we brought through human rebellion. “This better world really did exist,” they say, “and was designed for flourishing and intimacy with God. However, we rejected God, put ourselves in the place of God and as a result damaged the world.”
Yet here we see the good and the bad of contextualization. The good is seen in how they unpack words like idolatry with the helpful phrase “putting ourselves in the place of God.” The bad is in what they do not say. After all, why is it a problem that we put ourselves in God’s place? Is it bad simply because it wrecks our world or because it breaks his law, lies about his glory, and earns his just wrath? In an attempt to simplify the presentation many crucial questions go unanswered.
In world three we learn, that
Instead of leaving us in our brokenness God sends Jesus to be like us, to die on the cross and to rise from the dead. In this Jesus identifies with us, owns our judgment we deserve for damaging the world, and releases his power to restore the world for better.
Again, all of these statements are true but they are dangerously reductionistic. How does Jesus become like us? After all, we are the ones who messed everything up (see world 2). Did he contribute to this? Why did he have to die on the cross? How does this intersect with how I have “damaged” the world?
The fourth world is the invitation to join Jesus and his community in healing the world. In order to do this we must do three things:
(1) Identify with Jesus; believe that his death and resurrection broke the corruption in the world and in our hearts.
(2) Own our responsibility for the damage and the scarring in this world.
(3) Overcome by choosing to follow Jesus. Jesus does not leave us alone; he gives us himself, the Holy Spirit, and his people to go together and follow people.
These things are not untrue but they are, in my view, unclear. Following Jesus is reduced to becoming a conduit of healing. If we have not explained who God is, what sin is, and how the reconciliation is exclusively achieved through the cross of Christ, then we are not being totally forthright. At some point Christians have to agree that the gospel has irreducible components. These categories need to be developed and explained; they cannot be glossed over and certainly cannot be replaced with vague phrases like becoming “a conduit of healing.”
Another point that I found troubling in reviewing the material was the sheer lack of Bible. The videos I reviewed were curiously devoid of any mention of Scripture, even in passing. Yet the Bible should feature prominently in our evangelism. After all, it is the word of Christ that brings faith (Rom. 10:17).
BOTTOM LINE
What’s my bottom line?
I appreciate and even applaud the four worlds, the drawings, and the goal of listening to people’s stories in order to show how they fit within the big picture of God’s story. However, in setting out to do this, we must exercise great care about what we say, not just how we say it.
While it’s set in the Bible’s storyline, the New World Gospel Presentation is so user-friendly that it is simply not Christian enough. I would imagine that Roman Catholics and even Mormons could use this material within their doctrinal framework without violating their convictions. While helpful in some points, the New World Gospel Presentation simply lacks the main ingredients of the gospel. I would not recommend this program for use in your church.
[1] http://evangelism.intervarsity.org/how/gospel-outline/new-world-gospel-presentation; accessed 8/5/13.
Erik Raymond is the pastor of Emmaus Bible Church in Omaha, Nebraska.
Evangelism Tool Review: Two Ways to Live [9Marks Blog: Building Healthy Churches] 2013-09-04 00:01

Every Christian should know the gospel and be able to present it to others (1 Pet. 3:15). Now, our circumstances, personalities, and gifts will vary hugely. Nonetheless, if you are a follower of Jesus, you should know the central message of Christianity, and be able to articulate it faithfully and clearly. Two Ways to Live by Matthias Media is an excellent resource to help you do just that.
PRESENTATION
Two Ways to Live summarizes the message of Christianity in six steps, following the logic and storyline of the Bible.
First, this is an excellent gospel presentation. Most importantly, it is faithful to Scripture. This is the good news of Jesus Christ, as foretold by the Old Testament and proclaimed by the New.
Second, it’s easy to remember. It is two points longer than the “God, Man, Christ, Response” outline that I’m most familiar with. But the additional points are simply an expansion on Man (points 2 and 3) and Christ (points 4 and 5). In a culture resistant to the idea of sin and judgment, it’s good to give a little more time to the reality and consequence of our rebellion. And given how easily we can treat the resurrection as an afterthought, it’s helpful to have a separate discussion of its significance, both in our salvation and in the coming judgment.
Third, as people are increasingly illiterate when it comes to the Bible, Two Ways to Live presents the gospel in language and ideas that are understandable. By talking about God as king, sin as rebellion, judgment as being cut off from God’s goodness and punished by God, repentance and faith as submitting and relying, Two Ways to Live presents the gospel without using too many Christian-y terms, but without watering it down either.
RELATED RESOURCES
So the content of TWTL is faithful and useful. Even more useful, there are three categories of resources that go with it:
In other words, there is no shortage of ways you can use this in your discipleship and your evangelism.
BOTTOM LINE
Bottom line? Use Two Ways to Live! We want to be prepared to give the reason for the hope that we have to anyone who asks. Two Ways To Live is a great place to start.
Geoff Chang is an associate pastor of Hinson Baptist Church in Portland, Oregon.
Evangelism Tool Review: The Story [9Marks Blog: Building Healthy Churches] 2013-09-03 13:02

The Story is a gospel presentation tool designed by SpreadTruth.com. It is a popular tool for sharing the gospel; half a million people have viewed the gospel presentation online and The Story ESV Bible was published by Crossway in 2013. The Story can be accessed online (at http://viewthestory.com) through an app or via traditional printed tracts.
But is it any good? Well, leave it to us here at 9Marks to criticize the way other people share the gospel. I’ll discuss a few strengths and a few eyebrow-raising issues, then give a bottom line.
STRENGTHS
1. Biblical theology. The Story begins at creation and works its way through to the consummation of all things. This is a very good thing. The gospel is a message that comes to us with a context. The Story does a great job with that context.
2. Penal substitution. The Story gets the heart of the gospel right: on the cross Jesus bore our guilt and the wrath that our sin deserved. A lot of gospel presentations develop “alligator arms” at this point, so it’s nice to see that clearly presented.
EYEBROW-RAISERS
1. Visuals. This is the least important quibble, but the pages of the online version of The Story switch back and forth between visual styles in a way that is distracting. Sometimes the colors and bright and the font is crisp, at other points it feels gothic and grim in a way that doesn’t aesthetically connect with what came before it. Just a small peeve, but it feels like a missed opportunity to create something that is visually arresting.
2. Soft-pedaling condemnation. The way The Story talks about the consequences of sin is less than the full truth. The ultimate consequence of sin, it says, is “eternal separation from a loving God, in terrible misery and unhappiness.” And it refers to hell as a “painful separation.” That’s just not good enough. The Bible teaches that sinners aren’t merely separated from God; they are under his wrath. I fear that by soft-pedaling condemnation, The Story’s presentation of the gospel sells God’s holiness short.
BOTTOM LINE
While I’m grateful that The Story gets the heart of the gospel right and frames the gospel in the biblical narrative, I won’t be switching over to use it. Instead, I’d recommend Jesus. Who, Why… So What? or Two Ways to Live. Both of those resources do a better job communicating the gospel with sharp edges intact.
Mike McKinley is the senior pastor of Sterling Park Baptist Church in Sterling, Virginia, and is the author, most recently, of The Devil Made Me Do It (Good Book Company, 2013).
Connecting Evangelism and Church [9Marks Blog: Building Healthy Churches] 2013-08-29 00:01

Is evangelism an individual sport or a team sport? Really, it’s both.
Think of fishing. There are times you might saunter down to the dock by your lonesome, dangle your feet off the side, and cast in a line. But ask the men on an ocean trawler what it takes to haul a ton of wriggling mackerel out of writhing seawaters. They desperately need one another.
The fishing analogy does not say everything we would want to say about the relationship between evangelism and the local church, but it’s biblical, and it’s a start. Jesus told the disciples to follow him, that he would make them fishers of men, and then he sent them out two by two to preach that people should repent (Mark 1:17; 6:7, 13). Like fishermen on a trawler, we need the church to do the work of evangelism.
Yet there’s a bigger picture to see in relating evangelism and the church. Think of the first chapters of Acts, where the apostles proclaimed the resurrection, and behind them was the church, living together and sharing everything in common, “praising God and enjoying the favor of the people” (2:47; also 5:13). Somehow, the life of the church, sitting there as a backdrop to the proclamation of the gospel, served as a witness to the gospel. It caused many in Jerusalem to view the saints with favor, and it seemed to lead to more conversions.
Was it these early days in Jerusalem that Peter had in mind when he later described the church as a people, a priesthood, and a nation “that you may declare the praises of him” who called us out of darkness, and to live such good lives that pagans would see our good deeds “and glorify God” (1 Peter 2:9, 12)?
In both the early chapters of Acts and 1 Peter 2, one gets the feel of the church as a beehive, a buzzing ball of honey-making sweetness, swarming with the comings and going of busy worker bees. The hive is essential to the individual bee’s work, and part of the work. What might all this say about the relationship between evangelism and a church?
No analogy goes all the way and captures everything. Let’s see if we might sum up the relationship between the church and evangelism in the Bible in four systematic statements, and then ask what practical lessons follow for churches.
1. Evangelism Points to God, Not to the Church
If you were trying to convince someone to join your club, you would point to all the benefits of the club: the fun members have with one another, the annual table tennis tournament, and so forth. This is not how it works with evangelism and the church.
Evangelism points to God, not to the church. That’s the first statement.
Paul tells the Corinthians that Christ had given him (and them) a “ministry of reconciliation” and a “a message of reconciliation.” He (and they) were “Christ’s ambassadors, as though God were making his appeal through us.” And this message of reconciliation is simple: “Be reconciled to God” (2 Cor. 5:18-21).
The evangelist’s good news is not, “Be reconciled to other people,” even though the good news will lead to being so reconciled. Rather, the evangelist’s good news is how a person can be reconciled to God. Everything else flows from this.
2. The Church is One Outcome of Evangelism
By the same token, the first hoped for outcome of evangelism is reconciliation with God. But there is a second hoped for outcome: reconciliation with the people of God, the church.
If your doctrine of conversion is missing the corporate element, it’s missing an essential piece of the whole. A covenant head must have a covenant people. Our corporate unity in Christ is not just an implication of conversion, it’s part of the very thing. Being reconciled to God’s people is distinct from but inseparable from being reconciled to God (see my “The Corporate Component of Conversion”).
All this is put on display wonderfully in Ephesians 2. Verses 1 to 10 explain forgiveness and our vertical reconciliation with God: “By grace you have been saved.” Verses 11 to 22 then present the horizontal: “For he himself is our peace, who has made us both one and has broken down in his flesh the dividing wall of hostility” (v. 14). Notice that the activity of verse 14 is in the past tense. Christ has already made Jew and Gentile one. It’s what they are because God has done it, and God did it in precisely the same place he accomplished the vertical reconciliation—in the cross of Christ (see also Eph. 4:1-6).
In short, we are saved into a people.
The early chapters of Acts demonstrate what this looks like in practice: “Those who accepted his message were baptized, and about three thousand were added to their number that day” (Acts 2:41; see also 2:47; 4:4; 6:7). People trust in Christ and are added to “the number” of the church in Jerusalem. They are counted. Their name gets added. If they had had cameras, a photo no doubt would have gone into the church directory!
The converted life is congregationally shaped. Christians belong in churches, and so this is where the evangelist will send people.
3. Evangelism is the Work of the Church
Third, evangelism is the work of the church. Once a person is reconciled to God and (therefore) to God’s people, he or she gains a new job: sharing the gospel with others. “Follow me, and I will make you fishers of men,” said Jesus (Mark 1:17; also, Matt. 28:19). Every Christian and church member, in other words, is charged with sharing the gospel (see Timothy Beougher, “Must Every Christian Evangelize?”).
The first chapters of Acts emphasize the preaching of the Apostles, but when persecution broke out in Jerusalem and the church scattered, “Those who had been scattered preached the gospel wherever they went” (Acts 8:4).
Local churches exist to worship God and share the good news of Jesus Christ. This is why the teachers teach and the members learn. In fact, Jesus gives the so-called evangelists, pastors, and teachers to the church to equip them to do ministry (Eph. 4:11f), a ministry that surely includes evangelism.
We work together to haul in the fish.
4. The Church Is an Apologetic in Evangelism
The life of a converted people, grouped together in congregations, should also commend the gospel that saved them. “Gospel doctrine,” Ray Ortlund has written, “creates a gospel culture.” And that culture, embodied in our churches, should be attractive to outsiders, at least to some (see 2 Cor. 2:15-16).
This brings us back to the picture of the church as a humming, honey-filled beehive. We see this in Acts and 1 Peter 2. We also see it in Matthew 5, when Jesus talks about the church being salt and light (vv. 13-16). And it’s remarkably pictured in John 13, where Jesus observes, “Just as I have loved you, you also are to love one another. By this all people will know that you are my disciples, if you have love for one another” (vv. 34-35).
Our good deeds toward outsiders and our love for our fellow church members points neighbors and colleagues to Jesus!
All that to say, the local church is an apologetic in evangelism. The life of the church argues for the gospel. Believers living with one another testifies to the power of God in salvation. As we sit under the preaching of God’s Word week after week, and as the Spirit conforms us to the image of the Son little by little, we exemplify what the gospel can do to us as individuals and as a people.
Slowly, we are becoming the new humanity, following after the one who is the firstborn of the new creation (Col. 1:15). And this new humanity serves as a wonderful backdrop or billboard in our evangelism. It offers a contrast culture to the cultures of this world.
PRACTICAL TAKE-AWAYS
What are some practical lessons we can take from these four systematic principles? Often, pastors try to strengthen a church’s evangelistic ministry by exhorting people to share the gospel. Surely that’s one piece. But it’s also critical to grow the church as a contrast culture, which acts as this attractive backdrop for evangelism.
1) Evangelism should lead to baptism and membership. Churches should not evangelize and then leave new converts out on their own. Nor should they evangelize, baptize, and then, maybe, someday, get around to bringing someone into church membership. Except for exceptional circumstances (e.g., Ethiopian eunuch), churches should do what the church in Jerusalem did: baptize people into their number (Acts 2:41). Baptism, after all, is the corporate and authorized sign by which a church formally affirms a person as a believer. That affirmation should then be protected and nurtured by the ongoing oversight given through membership and the Lord’s Supper. We don’t leave new hatchlings outside of the nest, but bring them inside.
2) Teach members to integrate their lives with one another. In order to strengthen a church’s apologetic power, members should constantly be reminded through the teaching of the word and the celebration of the Lord’s Supper that we are one body (e.g. 1 Cor. 10:16-17; 1 Cor. 12). Hardly a Sunday should go by when members are not reminded to build relationships with one another so that they might encourage, build up, strengthen, speak truth, warn, and love one another (e.g. Rom. 12:9-13; Eph. 4:11-32). They should be exhorted to show hospitality (Rom. 12:13; 1 Peter 4:9). All this creates an attractive witness for the gospel.
3) Teach members to sacrifice for one another. Even more specifically, Christians should think about how they might better sacrifice for one another, financially and otherwise (e.g. Acts 2:42-46; 2 Cor. 8-9; 1 Peter 4:10). In a consumeristic nation, especially, the example of shared generosity among believers presents a powerful contrast culture. Remember, Jesus told Christians to love one another as he has loved us (John 13:34)—a sacrificial love if there ever was one.
4) Practice church discipline. Christian hypocrites and heretics in our midst compromise the witness of the church. When the church members in a community are known as liars, backbiters, and adulterers, that church’s evangelistic work will not go so well. That’s not to say that a church should discipline every saint who still struggles with sinning in their midst. Then there would be no church left. Rather, churches should confront and discipline unrepentant sin. This serves, ironically, to evangelize the unrepentant member (see 1 Cor. 5:4), as well as a church’s city more broadly (see 1 Cor. 5:1-2).
5) Equip members to share the gospel. Church leaders should look for various ways to make sure every member can explain the basics of the faith. This can be done from the pulpit, the Sunday School classroom, the membership interview, and elsewhere (see Kevin McKay, “Overcoming Objections to Evangelism”).
6) Encourage members to live lives that bless outsiders. Church members, hopefully, are known as kind, friendly, and quick to lend a hand. We should be quick to jump in with a rake to help clear the neighbor’s leaves, quick to offer help to an office-mate, quick to defend a victim of abuse, quick to work hard at preserving the jobs of hard-working employees in difficult times, quick to bless in all sorts of ways. Good deeds should adorn our evangelistic words.
7) Invite people into formal and informal gatherings of the church. Countless stories could be given of how non-believers heard the gospel and then watched the church in motion, both in formal or informal gatherings, and then came to faith. The church’s life together compelled them. It pointed to something they had never known in their family, school, or workplace. In other words, inviting outsiders into the life of the church surely must constitute one part of our evangelism.
8) Set the example in evangelism. Wherever a church’s elders are known for their evangelism, you can expect to find an evangelistic church. Where the elders don’t, you won’t.
9) Feature evangelism and conversion stories. Church leaders should pepper stories of evangelistic encounters into their sermons and lessions. Church members should share prayer requests for evangelistic opportunities. Baptismal candidates should be given the chance to share their conversion experience. Things like these all help to make evangelism a "normal" part of the Christian life and the church experience.
10) Brag about your church. The apostle Paul sometimes boasted about his churches as a way of boasting about Christ (see 2 Cor. 9:2; 2 Thes. 1:4; cf. Phil. 2:16). Christians, likewise, should look for ways to speak positively and gratefully—not obnoxiously or pridefully—about their churches around non-Christian friends. When a colleague asks about the weekend, mention how your church gave your wife a wonderful baby shower. Mention something encouraging the preacher said on Sunday. Mention the work your congregation is doing at the shelter when the subject of homelessness comes up. Doing this well, no doubt, takes practice.
CONCLUSION
Rightly relating church and evangelism in our understanding and practice requires more than exhorting people to evangelize. It requires attending to matters of polity and governance, membership and discipline. It requires building a healthy church that sits under God’s preached Word, and knows what God has tasked the church to do.
It requires godly leaders who teach and set the example. And it requires members who love Jesus and increasingly can’t help but sing the praises of him who brought them from death to life—inside and outside the church building.
Jonathan Leeman, an elder of Capitol Hill Baptist Church and the editorial director of 9Marks, is the author of several books on the local church. You can follow him on Twitter.
Must Every Christian Evangelize? [9Marks Blog: Building Healthy Churches] 2013-08-27 00:01
Does the Bible require every Christian to evangelize? How we answer this question will radically impact the shape of pastoral ministry and daily discipleship, so it’s imperative to answer it correctly.
ANSWERING TWO COMMON ARGUMENTS
The scriptural answer is “yes.” But I have encountered two main reasons for why some argue the answer is “no.”
1. The Great Commission was only given to the apostles and therefore does not apply to us today.
First, some argue that the Great Commission was only given to the apostles and therefore does not apply to us today. While it is true that contextually the Great Commission (Matt. 28:18-20) was given to the apostles, it was not only for the apostles. The command “teaching them to observe all that I have commanded you” certainly includes the command to make disciples. D.A. Carson notes that the Great Commission does not record Jesus saying to the apostles, “. . . teaching them to obey everything I have commanded you, except for this commandment to make disciples. Keep their grubby hands off that one, since it belongs only to you, my dear apostles.”[1]
What had Jesus commanded the apostles? Among many other things, he commanded them to preach the gospel to the whole creation. So this command of Jesus given to the apostles also applies to every believer today. In addition, should we try to limit Jesus’ promise “I am with you always, to the end of the age,” as only applying to the apostles, or does it apply to us today? Certainly it applies to us today!
2. Since only some people have the “gift of evangelism,” not everyone is obligated to witness.
Second, some claim that since only some people have the “gift of evangelism,” not everyone is obligated to witness. Space prohibits a full discussion on the topic of “the gift of evangelism,” but a few observations are in order.
First, evangelism is not recorded in the common spiritual gifts listings in Scripture; instead, the office of evangelist is mentioned in Ephesians 4:11. Some (myself included) question whether “evangelism” should be seen as a distinct spiritual gift, such as giving, serving, and so on.
In addition, even if evangelism is a spiritual gift, it is also a command for all believers, just like giving, serving, and so on. Not having “the gift of evangelism” does not excuse a believer from his or her call to share Christ with others.
FOUR BIBLICAL REASONS WHY EVERY CHRISTIAN SHOULD EVANGELIZE
Does Scripture mandate that every believer should evangelize? I argue “yes,” for the following four reasons.
1. The commands to witness are given to all followers of Christ
First, the commands to witness are given to all followers of Christ. Acts 1:8, for example, reads, “But you will receive power when the Holy Spirit has come upon you, and you will be my witnesses in Jerusalem and in all Judea and Samaria, and to the end of the earth.” This verse gives a command from the risen Lord to all his followers. As John Stott argues, “We can no more restrict the command to witness than we can restrict the promise of the Spirit.”[2]
In writing to the Corinthian believers, Paul maintained,
All this is from God, who through Christ reconciled us to himself and gave us the ministry of reconciliation; that is, in Christ God was reconciling the world to himself, not counting their trespasses against them, and entrusting to us the message of reconciliation. Therefore, we are ambassadors for Christ, God making his appeal through us. We implore you on behalf of Christ, be reconciled to God. (2 Cor. 5:18-20)
It’s not only apostles that have the ministry of reconciliation and the role of Christ’s ambassadors—all believers do! Other verses that reflect on this ministry of witness for all believers include Matthew 5:14-16, 1 Peter 3:15, Philippians 2:14-16, Colossians 4:5-6 and 1 Peter 2:9.[3]
2. The example of “ordinary believers” in the early church
Second, consider the example of “ordinary believers” in the early church. As we follow the storyline of the early church it is obvious that the apostles sought to evangelize and disciple others. But we see ordinary believers sharing the gospel as well.
Following the stoning of Stephen we read in Acts 8:1, “And there arose on that day a great persecution against the church in Jerusalem, and they were all scattered throughout the regions of Judea and Samaria, except the apostles.” And what did those ordinary believers do? Acts 8:4 tells us: “Now those who were scattered went about preaching (euangelizomenoi) the word.” They went about sharing the gospel with others.
Noted historian Kenneth Scott Latourette makes this observation about the spread of the gospel:
The chief agents in the expansion of Christianity appear not to have been those who made it a profession or a major part of their occupation, but men and women who earned their livelihood in some purely secular manner and spoke of their faith to those whom they met in this natural fashion.[4]
3. The stewardship the gospel imposes on us.
Third, consider the stewardship the gospel imposes on us. Jesus reminds us, “Everyone to whom much was given, of him much will be required” (Luke 12:48). We have been given no greater gift than the gospel, and we have no greater stewardship than to share that message of good news with others. Paul expresses it well in 2 Corinthians 5:14: “for the love of Christ controls us.”
4. The “work of ministry” in Ephesians 4.
Finally, consider what Paul calls “the work of ministry” in Ephesians 4. In this chapter Paul notes different offices in the church (apostles, prophets, evangelists, shepherds and teachers). He declares part of the reason God “gifts” the church with such leaders is so they will “equip the saints for the work of ministry, for building up the body of Christ” (Eph. 4:12). And we should certainly include evangelism in “the work of ministry.”
Ephesians 4 raises a challenge for pastors: Are we training our people to do evangelism? Are we setting an example for them in our own personal evangelism? Some people run from the idea of evangelism because they assume it means they must be obnoxious and pushy. There are many approaches to sharing the gospel. The only fixed method is the message: telling others about the gospel of Jesus Christ.
LEAD BY EXHORTATION AND ESPECIALLY EXAMPLE
Pastors, we can say to our people with confidence, “you are called to be a witness for Christ in both word and deed.” As leaders, let us challenge other believers not only with our exhortations but also with our example.[5] And let us take great confidence in the gospel, “for it is the power of God for salvation to everyone who believes, to the Jew first and also to the Greek” (Rom. 1:16).
Tim Beougher has served as the Billy Graham Professor of Evangelism and Associate Dean of the Billy Graham School at the Southern Baptist Theological Seminary since 1996. He is the author of numerous works, including Richard Baxter and Conversion (Christian Focus, 2007) and Overcoming Walls to Witnessing (BGEA, 1993).
[1] D.A. Carson, “Ongoing Imperative for World Mission,” in The Great Commission: Evangelicals and the History of World Missions, edited by Martin I. Klauber and Scott M. Manetsch (Broadman & Holman, 2008), 179.
[2] John R.W. Stott, Our Guilty Silence (Inter-Varsity Press, 1967), 58.
[3] While the context of 1 Peter 3:15 is what can be called “passive evangelism” (responding to a question that an unbeliever asks), this command is clearly given to all believers “to be ready” to answer when asked.
[4] Kenneth Scott Latourette, A History of the Expansion of Christianity (Harper & Brothers, 1937), 1:116.
[5] Among the many helpful resources for personal evangelism, I highly recommend: Will Metzger, Tell the Truth; Mark Dever, The Gospel & Personal Evangelism; and J.I. Packer, Evangelism and the Sovereignty of God.
Christianity and the Dark Side—What about Halloween? [AlbertMohler.com » Blog] 2013-10-30 03:53
Over a hundred years ago, the great Dutch theologian Herman Bavinck predicted that the 20th century would “witness a gigantic conflict of spirits.” His prediction turned out to be an understatement, and this great conflict continues into the 21st century.
The issue of Halloween presses itself annually upon the Christian conscience. Acutely aware of dangers new and old, many Christian parents choose to withdraw their children from the holiday altogether. Others choose to follow a strategic battle plan for engagement with the holiday. Still others have gone further, seeking to convert Halloween into an evangelistic opportunity. Is Halloween really that significant?
Well, Halloween is a big deal in the marketplace. Halloween is surpassed only by Christmas in terms of economic activity. Reporting in 2007, David J. Skal estimated: “Precise figures are difficult to determine, but the annual economic impact of Halloween is now somewhere between 4 billion and 6 billion dollars depending on the number and kinds of industries one includes in the calculations.” As of 2012, that total exceeded $8 billion.
Furthermore, historian Nicholas Rogers claims:
Halloween is currently the second most important party night in North America. In terms of its retail potential, it is second only to Christmas. This commercialism fortifies its significance as a time of public license, a custom-designed opportunity to have a blast. Regardless of its spiritual complications, Halloween is big business.
Rogers and Skal have each produced books dealing with the origin and significance of Halloween. Nicholas Rogers is author of Halloween: From Pagan Ritual to Party Night. Professor of History at York University in Canada, Rogers has written a celebration of Halloween as a transgressive holiday that allows the bizarre and elements from the dark side to enter the mainstream. Skal, a specialist on the culture of Hollywood, has written Death Makes a Holiday: A Cultural History of Halloween. Skal’s approach is more dispassionate and focused on entertainment, looking at the cultural impact of Halloween in the rise of horror movies and the nation’s fascination with violence.
The pagan roots of Halloween are well documented. The holiday is rooted in the Celtic festival of Samhain, which came at summer’s end. As Rogers explains, “Paired with the feast of Beltane, which celebrated the life-generating powers of the sun, Samhain beckoned to winter and the dark nights ahead.” Scholars dispute whether Samhain was celebrated as a festival of the dead, but the pagan roots of the festival are indisputable. Questions of human and animal sacrifices and various occultic sexual practices continue as issues of debate, but the reality of the celebration as an occultic festival focused on the changing of seasons undoubtedly involved practices pointing to winter as a season of death.
As Rogers comments: “In fact, the pagan origins of Halloween generally flow not from this sacrificial evidence, but from a different set of symbolic practices. These revolve around the notion of Samhain as a festival of the dead and as a time of supernatural intensity heralding the onset of winter.
How should Christians respond to this pagan background? Harold L. Myra of Christianity Today argues that these pagan roots were well known to Christians of the past:
More than a thousand years ago Christians confronted pagan rites appeasing the lord of death and evil spirits. Halloween’s unsavory beginnings preceded Christ’s birth when the druids, in what is now Britain and France, observed the end of summer with sacrifices to the gods. It was the beginning of the Celtic year and they believed Samhain, the lord of death, sent evil spirits abroad to attack humans, who could escape only by assuming disguises and looking like evil spirits themselves.
Thus, the custom of wearing costumes, especially costumes imitating evil spirits, is rooted in the Celtic pagan culture. As Myra summarizes, “Most of our Halloween practices can be traced back to the old pagan rites and superstitions.”
The complications of Halloween go far beyond its pagan roots, however. In modern culture, Halloween has become not only a commercial holiday, but a season of cultural fascination with evil and the demonic. Even as the society has pressed the limits on issues such as sexuality, the culture’s confrontation with the “dark side” has also pushed far beyond boundaries honored in the past.
As David J. Skal makes clear, the modern concept of Halloween is inseparable from the portrayal of the holiday presented by Hollywood. As Skal comments, “The Halloween machine turns the world upside down. One’s identity can be discarded with impunity. Men dress as women, and vice versa. Authority can be mocked and circumvented, and, most important, graves open and the departed return.”
This is the kind of material that keeps Hollywood in business. “Few holidays have a cinematic potential that equals Halloween’s,” comments Skal. “Visually, the subject is unparalleled, if only considered in terms of costume design and art direction. Dramatically, Halloween’s ancient roots evoke dark and melodramatic themes, ripe for transformation into film’s language of shadow and light.”
But television’s “It’s the Great Pumpkin, Charlie Brown” (which debuted in 1966) has given way to Hollywood’s “Halloween” series and the rise of violent “slasher” films. Bela Lugosi and Boris Karloff have been replaced by Michael Myers and Freddy Kruger.
This fascination with the occult comes as America has been sliding into post-Christian secularism. While the courts remove all theistic references from America’s public square, the void is being filled with a pervasive fascination with evil, paganism, and new forms of occultism.
In addition to all this, Halloween has become downright dangerous in many neighborhoods. Scares about razor blades hidden in apples and poisoned candy have spread across the nation in recurring cycles. For most parents, the greater fear is the encounter with occultic symbols and the society’s fascination with moral darkness.
For this reason, many families withdraw from the holiday completely. Their children do not go trick-or-treating, they wear no costumes, and they attend no parties related to the holiday. Some churches have organized alternative festivals, capitalizing on the holiday opportunity, but turning the event away from pagan roots and the fascination with evil spirits. For others, the holiday presents no special challenges at all.
These Christians argue that the pagan roots of Halloween are no more significant than the pagan origins of Christmas and other church festivals. Without doubt, the church has progressively Christianized the calendar, seizing secular and pagan holidays as opportunities for Christian witness and celebration. Anderson M. Rearick, III argues that Christians should not surrender the holiday. As he relates, “I am reluctant to give up what was one of the highlights of my childhood calendar to the Great Imposter and Chief of Liars for no reason except that some of his servants claim it as his.”
Nevertheless, the issue is a bit more complicated than that. While affirming that make-believe and imagination are part and parcel of God’s gift of imagination, Christians should still be very concerned about the focus of that imagination and creativity. Arguing against Halloween is not equivalent to arguing against Christmas. The old church festival of “All Hallow’s Eve” is by no means as universally understood among Christians as the celebration of the incarnation at Christmas.
Christian parents should make careful decisions based on a biblically-informed Christian conscience. Some Halloween practices are clearly out of bounds. Others may be strategically transformed, but this takes hard work and may meet with mixed success.
The coming of Halloween is a good time for Christians to remember that evil spirits are real and that the Devil will seize every opportunity to trumpet his own celebrity. Perhaps the best response to the Devil at Halloween is that offered by Martin Luther, the great Reformer: “The best way to drive out the devil, if he will not yield to texts of Scripture, is to jeer and flout him for he cannot bear scorn.”
On October 31, 1517, Martin Luther began the Reformation with a declaration that the church must be recalled to the authority of God’s Word and the purity of biblical doctrine. With this in mind, the best Christian response to Halloween might be to scorn the Devil and then pray for the Reformation of Christ’s church on earth. Let’s put the dark side on the defensive.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/albertmohler
A Clear and Present Danger: Religious Liberty, Marriage, and the Family in the Late Modern Age — An Address at Brigham Young University [AlbertMohler.com » Blog] 2013-10-21 15:35
An address delivered at Brigham Young University by Dr. R. Albert Mohler, Jr., President of The Southern Baptist Theological Seminary on Monday, October 21, 2013.
I deeply appreciate your invitation to speak at Brigham Young University and to address the faculty at this greatly respected center of learning. I am so glad to be on this campus, filled with so many gracious people, such admirable students, and so many committed scholars on the faculty. To many people, shaped in their worldview by the modern age and its constant mandate to accommodate, it will seem very odd that a Baptist theologian and seminary president would be invited to speak at the central institution of intellectual life among the Latter-Day Saints.
But here I am, and I am thankful for the invitation. The wonderfully prophetic Catholic novelist Flannery O’Connor rightly warned that we must “push back against the age as hard as it is pressing you.” I have come to Brigham Young University because I intend with you to push back against the modernist notion that only the accommodated can converse. There are those who sincerely believe that meaningful and respectful conversation can take place only among those who believe the least—that only those who believe the least and thus may disagree the least can engage one another in the kind of conversation that matters. I reject that notion, and I reject it forcefully. To paraphrase Dorothy Parker, that is the kind of idea that must not be cast aside lightly, but thrown with full force.
I come as a Christian theologian to speak explicitly and respectfully as a Christian—a Christian who defines Christianity only within the historic creeds and confessions of the Christian church and who comes as one committed to the Gospel of Jesus Christ and to the ancient and eternal Trinitarian faith of the Christian church. I have not come as less, and you know whom you have invited. I come knowing who you are—to an institution that stands as the most powerful intellectual center of the Latter-Day Saints, the most visible academic institution of Mormonism. You know who I am and what I believe. I know who you are and what you believe. It has been my great privilege to know friendship and share conversation with leaders of the LDS church, such as Elder Tom Perry, Elder Quentin Cook, and Elder Todd Christofferson. I am thankful for the collegiality extended by President Cecil Samuelson at this great university. We do not enjoy such friendship and constructive conversation in spite of our theological differences, but in light of them. This does not eliminate the possibility of conversation. To the contrary, this kind of convictional difference at the deepest level makes for the most important kind of conversation. This is why I am so thankful for your gracious invitation.
Our conversation comes in the context of a particularly interesting historical moment. We are living in times rightly, if awkwardly, described as the Late Modern Age. Just a decade or so ago, we spoke of the Postmodern Age, as if modernity had given way to something new. Like every new and self-declared epoch, the Postmodern Age was declared to be a form of liberation. Whereas the Modern Age announced itself as a secular liberation from a Christian authority that operated on claims of revelation, the Postmodern Age was proposed as a liberation from the great secular authorities of reason and rationality. The Postmodern Age, it was claimed, would liberate humanity by operating with an official incredulity toward all metanarratives. And yet, postmodern thought eventuated, as all intellectual movements must, in its own metanarrative. And then it passed away. We still speak of postmodern thinking, even as we speak rightly of postmodern architecture and postmodern art, but we are speaking, for the most part, of a movement that has given way and given up. In retrospect, the Postmodern Age was not a new age at all, only the alarm that announced the Late Modern Age. Modernity has not disappeared. It has only grown stronger, if also more complex.
The claim that humanity can only come into its own and overcome various invidious forms of discrimination by secular liberation is not new, but it is now mainstream. It is now so common to the cultures of Western societies that it need not be announced, and often is not noticed. Those born into the cultures of late modernity simply breathe these assumptions as they breathe the atmosphere, and their worldviews are radically realigned, even if their language retains elements of the old worldview.
Recent research demonstrates this clearly. The Pew Research Center has released a torrent of research underlining these trends. We are now told that one in five Americans is essentially secular—thoroughly secularized, with no religious affiliation at all. Even more revealing is the fact that one in three younger Americans under age 30 is so identified. If anything, anecdotal evidence and any sophisticated analysis of their worldviews indicate that these figures may be an underestimation. More recently, the researchers at Pew have revealed that American Judaism is being radically secularized, traced by evidence of skyrocketing intermarriage rates and very low estimates of religious belief. No belief system is immune or impervious to modernity.
There is plenty of evidence that the same phenomenon is at work among Roman Catholic young people. Among evangelical Christians, a frightening percentage of youth and “emerging adults” hold to what sociologist Christian Smith and his associates have called “Moralistic Therapeutic Deism,” a religion that bears no substantive resemblance to biblical Christianity.
The background to this great intellectual shift is the secularization of Western societies. Modernity has brought many cultural goods, but it has also, as predicted, brought a radical change in the way citizens of Western societies think, feel, relate, and reason. The Enlightenment’s liberation of reason at the expense of revelation was followed by a radical anti-supernaturalism that can scarcely be exaggerated. Looking at Europe and Great Britain, it is clear that the Modern Age has alienated an entire civilization from its Christian roots, along with Christian moral and intellectual commitments. This did not happen all at once, of course, though in nations such as France and Germany the change came very quickly. Scandinavian nations now register almost imperceptible levels of Christian belief. Increasingly, the same is true of both the Netherlands and Great Britain. Sociologists now speak openly of the death of Christian Britain—and the evidence of Christian decline is abundant.
Peter Berger, one of the founding fathers of the modern theory of secularization, has suggested that secularization should be better understood as pluralization: the presence of plural worldviews in proximity offering an array of intellectual and theological options. But the result is nearly the same. The world might be, as he says, “furiously religious,” but the modern world is not controlled by any coherent supernatural worldview.
Actually, Berger argues that secularization, in exactly the shape and form predicted by the prophets of secularization theory, did operate exactly according to plan in two social locations, western Europe and the American college and university campus.
In his important Massey Lectures delivered in 1991, Canadian philosopher Charles Taylor spoke of The Malaise of Modernity. The Modern Age, he argued, is marked by two great intellectual moves. The first intellectual move is a pervasive individualism. The second is the reduction of all public discourse to the authority of instrumental reason. The rise of modern individualism came at the cost of rejecting all other moral authorities. “Modern freedom was won by our breaking loose from older moral horizons,” Taylor explains. This required the toppling of all hierarchical authorities and their established moral orders. “People used to see themselves as part of a larger order,” he observed. “Modern freedom came about through the discrediting of such orders.”
The primacy of instrumental reason means the elimination of the old order and its specifically theological and teleological moral order. As Taylor explains:
No doubt sweeping away the old orders has immensely widened the scope of instrumental reason. Once society no longer has a sacred structure, once social arrangements and modes of action are no longer grounded in the order of things or the will of God, they are in a sense up for grabs. They can be redesigned with their consequences for the happiness or well-being of individuals as our goal.
More recently, Taylor has written the greatest work yet completed on the secular reality of our times. In A Secular Age, he describes three successive sets of intellectual conditions. In the first, associated with the Premodern Age of antiquity and the medieval synthesis, it was impossible not to believe. There was simply no intellectual alternative to theism in the West. There was no alternative set of explanations for the world and its operations, or for moral order. All that changed with the arrival of modernity. In the Modern Age it became possible not to believe. A secular alternative to Christian theism emerged as a real choice. As a matter of fact, choice now ruled the intellectual field. As Peter Berger famously observed decades ago, this is the “heretical imperative,” the imperative to choose. The third set of intellectual conditions is identified with late modernity and our own intellectual epoch. For most people living in the context of self-conscious late modernity, it is now impossible to believe.
This is a stunning intellectual and moral revolution. It defies exaggeration. We must recognize that it is far more pervasive than we might want to believe, for this intellectual revolution has changed the worldviews of even those who believe themselves to be opposed to it. If nothing else, many religious believers in modern societies now operate as theological and ideological consumers, constantly shopping for new intellectual clothing, even as they believe themselves to be traditional believers. Everything is now reduced to choice, and choice is, as Taylor reminds us, central to the moral project of late modernity, the project of individual authenticity.
As he explains this project: “There is a certain way of being human that is my way. I am called upon to live my life in this way, and not in imitation of anyone else’s. But this gives a new importance to being true to myself. If I am not, I miss the point of my life, I miss what being human is for me.”
The pressing question is this: can any sustainable moral order survive this scale of intellectual revolution? We hear in the today’s intellectual and ideological chorus the refrains of Karl Marx’s threat and promise as stated in The Communist Manifesto: “All that is solid melts into air.” The melting is everywhere around us.
The clearest demonstration of this monumental shift in morality and worldview is the revolution now underway with regard to marriage, the family, and human sexuality. Long ago, historians Will and Ariel Durant noted that sex is “a river of fire that must be banked and cooled by a hundred restraints.” The primary restraint has always been the institution of marriage itself, an institution that is inescapably heterosexual and based in the monogamous union of a man and a woman as husband and wife. In our times, the fires of sex and sexuality are increasingly unbanked and uncooled.
Similarly, Pitirim Sorokin, the founder of sociology at Harvard University, pointed to the regulation of sexuality as the essential first mark of civilization. According to Sorokin, civilization is possible only when marriage is normative and sexual conduct is censured outside of the marital relationship. Furthermore, Sorokin traced the rise and fall of civilizations and concluded that the weakening of marriage was a first sign of civilizational collapse.
We should note carefully that Sorokin made these arguments long before anything like homosexual marriage had been openly discussed, much less legislated. Sorokin’s insight was the realization that civilization requires men to take responsibility for their offspring. This was possible, he was convinced, only when marriage was held to be the unconditional expectation for sexual activity and procreation. Once individuals—especially males—are freed for sexual behavior outside of marriage, civilizational collapse becomes an inevitability. The weakening of marriage—even on heterosexual terms—has already brought a harvest of disaster to mothers and children abandoned in the name of sexual liberation.
We must note with honesty and candor that this moral revolution and the disestablishment of marriage did not begin with the demand of same-sex couples to marry. The subversion of marriage began within the context of the great intellectual shift of modernity. Marriage was redefined in terms of personal fulfillment rather than covenant obligation. Duty disappeared in the fog of demands for authenticity and the romanticized ideal of personal fulfillment. Marriage became merely a choice and then a personal expression. Companionate marriage was secularized and redefined solely in terms of erotic and romantic appeal—for so long as these might last.
In an important new book, Has Marriage for Love Failed?, French intellectual Pascal Bruckner ponders the secularly imponderable: has the romantic revolution of secular modernity led to human happiness? He thinks not.
He does clearly understand what modernity hath wrought: “Since the Enlightenment, marriage reforms have focused on three points: giving priority to feelings over obligation, doing away with the requirement of virginity, and making it easy for badly matched spouses to separate.”
Bruckner is right—devastatingly right. Note carefully that all three of these points require the secularization of the moral order and the marital contract. Feelings now rule, defined and projected at will. Virginity is, as Bruckner notes, an embarrassment for most moderns. Cohabitation is now the order of the day for young moderns, and for an astonishingly large percentage of their parents and grandparents. The young are indoctrinated into the morality of expressive sexuality and erotic fulfillment, with children hardly able to read force-fed the curriculum of “safe sex” and erotic experimentation. And, sadly, the divorce revolution has not only made marriage a tentative, if not temporary, condition, it has redefined marriage as nothing more than a public celebration of an essentially and non-negotiably individual act of self-expression.
As Barbara Defoe Whitehead has observed, expressive marriage was followed almost instantly by expressive divorce. Divorce, like marriage, now becomes an expected act of self-expression for moderns, complete with greeting cards, celebrations, and public announcements of new erotic and romantic availability.
Has this made moderns happier? This is where Pascal Bruckner is particularly helpful and insightful. Modern romantic love, he argues, simply cannot sustain marriage. He describes this reality as a “terrible absurdity.” Marriage has “become more difficult to endure since of all its roles it has retained only that of being a model of fulfillment. Because it wants to succeed at any cost, it is consumed with anxiety, fears the law of entropy, the aridity of slack periods.”
Add to this the realization that no one can now grow old and mellow. Ardor must continue and erotic fulfillment must rule, even into later decades of life and marriage. A revealing article appeared in the health pages of USA Today, announcing that Viagra is now a prominent factor in divorce among the middle-aged and older. As reporter Karen S. Peterson explained: “Nobody claims Viagra causes affairs or divorce. But increasingly, it is a factor in both, says Dominic Barbara, who heads a Manhattan law firm with 15 attorneys. In about one of every 15 or 20 new divorce cases, somebody mentions Viagra, he says.”
Heterosexuals did a very good job of undermining marriage before same-sex couples arrived with their demands. The marriage crisis is a moral crisis and it did not start with same-sex marriage, nor will it end there. The logic of same-sex marriage will not end with same-sex marriage. Once marriage can mean anything other than a heterosexual union, it can and must mean everything. It is just a matter of time.
Of course, one of the issues we must confront is the fact that marriage is a pre-political institution, recognized and solemnized throughout history by virtually every human culture and civilization. But we are living in an age in which everything is political and nothing is honored as pre-political. In the recent words of Justice Antonin Scalia, we are all now waiting for the other shoe (or shoes) to drop.
This has all been made possible by a breakdown in the immune system of human society—and this breakdown was no accident. Immunologists will explain that one of the wonders of human life is the fact that each of us receives from our mother an amazing array of defenses within our immune system. Throughout time, we develop further immunities to disease, or we grow sick and vulnerable. A severely compromised immune system leads to chronic disease, constant vulnerability, and potential death. If this is true for an individual, it is also true of a society or civilization.
We have forfeited our immunity against the breakdown of marriage, the family, and integrity of human sexuality. We can point to others who have been the prophets and agents of this self-injury to society, but we must recognize that we have all contributed to it, in so far as we have embraced essentially modern understandings of love, romance, liberty, personal autonomy, obligation, and authority. Furthermore, the separation of the conjugal union and openness to the gift of children has further undermined both our conscience and our credibility in the defense of marriage. We separated sex from marriage and marriage from reproduction. We sowed the seeds of the current confusion. At the very least, we did not address this confusion with sufficient moral clarity and credibility.
Marriage is the most basic unit of civilization. In fact, it is the basic molecular structure of human society. The redefinition of marriage will bring great human unhappiness. As Pascal Bruckner reminds us, this is true of heterosexual divorce. It promised happiness but has produced misery and brokenness. It declared itself to be liberation, but it imprisons all moderns in its penitentiary of idealized and unattainable romance and sexual fulfillment.
The family, as properly pre-political as marriage, is now the great laboratory for human social experimentation. Children are routinely sacrificed to the romantic whims and sexual demands of their parents, who may or may not be married, may or may not stay married, and may or may not include both a father and a mother at any point.
The epidemic of fatherlessness is well documented and no longer even denied, but there is no social consensus to address a phenomenon that has wrought incalculable human costs, both individually and socially.
A basic principle of Christian theology was once written into the moral immune system of Western civilization—what God commands and institutes is what leads to genuine human flourishing. Our civilization now lives in open revolt against that affirmation.
The moral revolution we are now witnessing on the issue of homosexuality is without precedent in human history in terms of its scale and velocity. We are not looking at a span of centuries, or even the length of one century. This revolution is taking place within a single human generation.
I would argue that no moral revolution on this scale has ever been experienced by a society that remained intact, even as no moral revolution of this velocity has yet been experienced. We can now see more clearly where this revolution began. It is virtually impossible to see where it ends.
But, for the first time in the experience of most Americans, the moral revolution revolving around marriage, the family, and human sexuality is now clearly becoming a religious liberty issue. The rights of parents to raise their children according to their most basic and fundamental theological and moral convictions are now at stake. Courts have ruled in some jurisdictions that parents cannot even “opt out” their children from sex education driven by moral revisionism. Legislatures in California and New Jersey have made it illegal for mental health professionals to tell minors that there is anything wrong with homosexual sexuality, orientation, or relationships. Parents are put on notice. How long will it be before the moral authority of the secular state is employed to allow children to “divorce” their parents? How long before the logic of sexual revolution and sexual self-expression leads to parents being told what they must allow and facilitate with their own children when it comes to sex, gender, and sexual orientation? The logic of moral change by legal coercion is already fully on display in many modern legal debates. How long will a respect for parental rights and religious liberty hold back the flooding river of this moral revolution?
Religious liberty is already severely compromised by modern political regimes that claim to be democratic and respectful of human rights. Given the shape of current arguments for sexual expression and liberty, religious institutions, especially schools, colleges, universities, welfare agencies, and benevolent ministries, are already under fire and under warning. Some have already been forced to make a decision: forfeit your convictions or forfeit your work. Some have chosen one, some the other. One way leads to an honorable extinction, the other to a dishonorable surrender. Both are violations of religious liberty.
The conflict of liberties we are now experiencing is unprecedented and ominous. Forced to choose between erotic liberty and religious liberty, many Americans would clearly sacrifice freedom of religion. How long will it be until many becomes most?
This is what brings me to Brigham Young University today. I am not here because I believe we are going to heaven together. I do not believe that. I believe that salvation comes only to those who believe and trust only in Christ and in his substitutionary atonement for salvation. I believe in justification by faith alone, in Christ alone. I love and respect you as friends, and as friends we would speak only what we believe to be true, especially on matters of eternal significance. We inhabit separate and irreconcilable theological worlds, made clear with respect to the doctrine of the Trinity. And yet here I am, and gladly so. We will speak to one another of what we most sincerely believe to be true, precisely because we love and respect one another.
I do not believe that we are going to heaven together, but I do believe we may go to jail together. I do not mean to exaggerate, but we are living in the shadow of a great moral revolution that we commonly believe will have grave and devastating human consequences. Your faith has held high the importance of marriage and family. Your theology requires such an affirmation, and it is lovingly lived out by millions of Mormon families. That is why I and my evangelical brothers and sisters are so glad to have Mormon neighbors. We stand together for the natural family, for natural marriage, for the integrity of sexuality within marriage alone, and for the hope of human flourishing.
The great Christian theologian Augustine, writing in the final years of the Roman Empire, reminded Christians that we live simultaneously as citizens of two cities: a heavenly city and an earthly city. The one is eternal, the other is passing. But the earthly city is also a city of God’s good pleasure and divine compassion. As a Christian, I am instructed by the Bible to work for the good and flourishing of this earthly city, even as I work to see as many as possible also become citizens of the heavenly city through faith in Christ Jesus.
In this city, I am honored to come among those who, though of a different faith, share common concerns and urgencies. I come as a Christian, and I come as one who is honored by your kind and gracious invitation. I come in the hope of much further conversations, conversations about urgencies both temporal and eternal. I am unashamed to stand with you in the defense of marriage and family and a vision of human sexual integrity. I am urgently ready to speak and act in your defense against threats to your religious liberty, even as you have shown equal readiness to speak and act in defense of mine. We share love for the family, love for marriage, love for the gift of children, love of liberty, and love of human society. We do so out of love and respect for each other.
That is why only those with the deepest beliefs, and even the deepest differences, can help each other against encroaching threats to religious liberty, marriage, and the family. I guess I am back to Flannery O’Connor again. We must push back against this age as hard as it is pressing against us. We had better press hard, for this age is pressing ever harder against us.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
Charles Taylor, The Malaise of Modernity, in the CBC Massey Lectures (Toronto: Anansi Press, 1991).
Charles Taylor, A Secular Age (Cambridge: The Belknap Press of Harvard University Press, 2007).
Pascal Bruckner, Has Marriage for Love Failed?, translated by Steven Rendall and Lisa Neal (Cambridge: Polity Press, 2013).
Peter Berger, “Secularization Falsified,” First Things, February 2008. http://www.firstthings.com/article/2008/01/002-secularization-falsified-1
Karen S. Peterson, “Till Viagra Do Us Part,” USA Today, Wednesday, March 21, 2001. http://usatoday30.usatoday.com/news/health/2001-03-21-viagra-wives.htm
Falling on Deaf Ears?—Why So Many Churches Hear So Little of the Bible [AlbertMohler.com » Blog] 2013-10-14 00:44
“It is well and good for the preacher to base his sermon on the Bible, but he better get to something relevant pretty quickly, or we start mentally to check out.” That stunningly clear sentence reflects one of the most amazing, tragic, and lamentable characteristics of contemporary Christianity: an impatience with the Word of God.
The sentence above comes from Mark Galli, senior managing editor of Christianity Today in an essay entitled, “Yawning at the Word.” In just a few hundred words, he captures the tragedy of a church increasingly impatient with and resistant to the reading and preaching of the Bible. We may wince when we read him relate his recent experiences, but we also recognize the ring of truth.
Galli was told to cut down on the biblical references in his sermon. “You’ll lose people,” the staff member warned. In a Bible study session on creation, the teacher was requested to come back the next Sunday prepared to take questions at the expense of reading the relevant scriptural texts on the doctrine. Cutting down on the number of Bible verses “would save time and, it was strongly implied, would better hold people’s interest.”
As Galli reflected, “Anyone who’s been in the preaching and teaching business knows these are not isolated examples but represent the larger reality.”
Indeed, in many churches there is very little reading of the Bible in worship, and sermons are marked by attention to the congregation’s concerns, not by an adequate attention to the biblical text. The exposition of the Bible has given way to the concerns, real or perceived, of the listeners. The authority of the Bible is swallowed up in the imposed authority of congregational concerns.
As Mark Galli notes:
It has been said to the point of boredom that we live in a narcissistic age, where we are wont to fixate on our needs, our wants, our wishes, and our hopes—at the expense of others and certainly at the expense of God. We do not like it when a teacher uses up the whole class time presenting her material, even if it is material from the Word of God. We want to be able to ask our questions about our concerns, otherwise we feel talked down to, or we feel the class is not relevant to our lives.
And Galli continues:
It is well and good for the preacher to base his sermon on the Bible, but he better get to something relevant pretty quickly, or we start mentally to check out. Don’t spend a lot of time in the Bible, we tell our preachers, but be sure to get to personal illustrations, examples from daily life, and most importantly, an application that we can use.
The fixation on our own sense of need and interest looms as the most significant factor in this marginalization and silencing of the Word. Individually, each human being in the room is an amalgam of wants, needs, intuitions, interests, and distractions. Corporately, the congregation is a mass of expectations, desperate hopes, consuming fears, and impatient urges. All of this adds up, unless countered by the authentic reading and preaching of the Word of God, to a form of group therapy, entertainment, and wasted time—if not worse.
Galli has this situation clearly in his sights when he asserts that many congregations expect the preacher to start from some text in the Bible, but then quickly move on “to things that really interest us.” Like . . . ourselves?
One of the earliest examples of what we would call the preaching of the Bible may well be found in Nehemiah 8:1-8 (ESV):
And all the people gathered as one man into the square before the Water Gate. And they told Ezra the scribe to bring the Book of the Law of Moses that the Lord had commanded Israel. So Ezra the priest brought the Law before the assembly, both men and women and all who could understand what they heard, on the first day of the seventh month. And he read from it facing the square before the Water Gate from early morning until midday, in the presence of the men and the women and those who could understand. And the ears of all the people were attentive to the Book of the Law. And Ezra the scribe stood on a wooden platform that they had made for the purpose. And beside him stood Mattithiah, Shema, Anaiah, Uriah, Hilkiah, and Maaseiah on his right hand, and Pedaiah, Mishael, Malchijah, Hashum, Hashbaddanah, Zechariah, and Meshullam on his left hand. And Ezra opened the book in the sight of all the people, for he was above all the people, and as he opened it all the people stood. And Ezra blessed the Lord, the great God, and all the people answered, “Amen, Amen,” lifting up their hands. And they bowed their heads and worshiped the Lord with their faces to the ground. Also Jeshua, Bani, Sherebiah, Jamin, Akkub, Shabbethai, Hodiah, Maaseiah, Kelita, Azariah, Jozabad, Hanan, Pelaiah, the Levites, helped the people to understand the Law, while the people remained in their places. They read from the book, from the Law of God, clearly, and they gave the sense, so that the people understood the reading.
Ezra and his companions stood on a platform before the congregation. They read the scriptural text clearly, and then explained the meaning of the Scripture to the people. The congregation received the Word humbly, while standing. The pattern is profoundly easy to understand: the Bible was read and explained and received.
As Hughes Oliphant Old comments, “This account of the reading of the Law indicates that already at the time of the writing of this text there was a considerable amount of ceremonial framing of the public reading of Scripture. This ceremonial framing is a witness to the authority of the Bible.” The reading and exposition took place in a context of worship as the people listened to the Word of God. The point of the sermon was simple: “to make clear the reading of the Scriptures.”
In many churches, there is almost no public reading of the Word of God. Worship is filled with music, but congregations seem disinterested in listening to the reading of the Bible. We are called to sing in worship, but the congregation cannot live only on the portions of Scripture that are woven into songs and hymns. Christians need the ministry of the Word as the Bible is read before the congregation such that God’s people—young and old, rich and poor, married and unmarried, sick and well—hear it together. The sermon is to consist of the exposition of the Word of God, powerfully and faithfully read, explained, and applied. It is not enough that the sermon take a biblical text as its starting point.
How can so many of today’s churches demonstrate what can only be described as an impatience with the Word of God? The biblical formula is clear: the neglect of the Word can only lead to disaster, disobedience, and death. God rescues his church from error, preserves his church in truth, and propels his church in witness only by his Word—not by congregational self-study.
In the end, an impatience with the Word of God can be explained only by an impatience with God. We all, both individually and congregationally, neglect God’s Word to our own ruin.
As Jesus himself declared, “He who has ears to hear, let him hear.”
Mark Galli, “Yawning at the Word,” Christianity Today [online edition], posted November 5, 2009. http://www.christianitytoday.com/ct/2009/novemberweb-only/144-41.0.html
Hughes Oliphant Old, The Reading and Preaching of the Scriptures in the Worship of the Christian Church, Volume 1: The Biblical Period (Grand Rapids: W.B. Eerdmans, 2007).
This commentary was originally posted Friday, February 19, 2010.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
The Expositors Summit 2013 at Southern Seminary—October 29-31
The goal of Christian preaching is nothing less than the glory of God in the Christlikeness of his people. For this reason, The Expositors Summit 2013 at The Southern Baptist Theological Seminary will seek to restore the primacy of expository preaching to the pulpits of local churches. Pastors, students, and all who love the Scriptures are invited to come hear Dr. R. Albert Mohler, Jr., H.B. Charles Jr., and Alistair Begg as the keynote speakers (along with other gifted leaders in various seminars).
How Pornography Works: It Hijacks the Male Brain [AlbertMohler.com » Blog] 2013-10-09 04:57
We are fast becoming a pornographic society. Over the course of the last decade, explicitly sexual images have crept into advertising, marketing, and virtually every niche of American life. This ambient pornography is now almost everywhere, from the local shopping mall to prime-time television.
By some estimations, the production and sale of explicit pornography now represents the seventh-largest industry in America. New videos and internet pages are produced each week, with the digital revolution bringing a host of new delivery systems. Every new digital platform becomes a marketing opportunity for the pornography industry.
To no one’s surprise, the vast majority of those who consume pornography are males. It is no trade secret that males are highly stimulated by visual images, whether still or video. That is not a new development, as ancient forms of pornography attest. What is new is all about access. Today’s men and boys are not looking at line pictures drawn on cave walls. They have almost instant access to countless forms of pornography in a myriad of formats.
But, even as technology has brought new avenues for the transmission of pornography, modern research also brings a new understanding of how pornography works in the male brain. While this research does nothing to reduce the moral culpability of males who consume pornography, it does help to explain how the habit becomes so addictive.
As William M. Struthers of Wheaton College explains, “Men seem to be wired in such a way that pornography hijacks the proper functioning of their brains and has a long-lasting effect on their thoughts and lives.”
Struthers is a psychologist with a background in neuroscience and a teaching concentration in the biological bases of human behavior. In Wired for Intimacy: How Pornography Hijacks the Male Brain, Struthers presents key insights from neuroscience that go a long way toward explaining why pornography is such a temptation for the male mind.
“The simplest explanation for why men view pornography (or solicit prostitutes) is that they are driven to seek out sexual intimacy,” he explains. The urge for sexual intimacy is God-given and essential to the male, he acknowledges, but it is easily misdirected. Men are tempted to seek “a shortcut to sexual pleasure via pornography,” and now find this shortcut easily accessed.
In a fallen world, pornography becomes more than a distraction and a distortion of God’s intention for human sexuality. It comes as an addictive poison.
Struthers explains:
Viewing pornography is not an emotionally or physiologically neutral experience. It is fundamentally different from looking at black and white photos of the Lincoln Memorial or taking in a color map of the provinces of Canada. Men are reflexively drawn to the content of pornographic material. As such, pornography has wide-reaching effects to energize a man toward intimacy. It is not a neutral stimulus. It draws us in. Porn is vicarious and voyeuristic at its core, but it is also something more. Porn is a whispered promise. It promises more sex, better sex, endless sex, sex on demand, more intense orgasms, experiences of transcendence.
Pornography “acts as a polydrug,” Struthers explains. As Dr. Patrick Carnes asserts, pornography is “a pathological relationship with a mood-altering experience.” Boredom and curiosity lead many boys and men into experiences that become more like drug addiction than is often admitted.
Why men rather than women? As Struthers explains, the male and female brains are wired differently. “A man’s brain is a sexual mosaic influenced by hormone levels in the womb and in puberty and molded by his psychological experience.” Over time, exposure to pornography takes a man or boy deeper along “a one-way neurological superhighway where a man’s mental life is over-sexualized and narrowed. This superhighway has countless on-ramps but very few off-ramps.
Pornography is “visually magnetic” to the male brain. Struthers presents a fascinating review of the neurobiology involved, with pleasure hormones becoming linked to and released by the experience of a male viewing pornographic images. These experiences with pornography and pleasure hormones create new patterns in the brain’s wiring, and repeated experiences formalize the rewiring.
And then, enough is never enough. “If I take the same dose of a drug over and over and my body begins to tolerate it, I will need to take a higher dose of the drug in order for it to have the same effect that it did with a lower dose the first time,” Struthers reminds us. So, the experience of viewing pornography and acting out on it creates a demand in the brain for more and more, just to achieve the same level of pleasure in the brain.
While men are stimulated by the ambient sexual images around them, explicit pornography increases the effect. Struthers compares this to the difference between traditional television and the new high definition technologies. Everything is more clear, more explicit, and more stimulating.
Struthers explains this with compelling force:
Something about pornography pulls and pushes at the male soul. The pull is easy to identify. The naked female form can be hypnotizing. A woman’s willingness to participate in a sexual act or expose her nakedness is alluring to men. The awareness of one’s own sexuality, the longing to know, to experience something as good wells up from deep within. An image begins to pick up steam the longer we look upon it. It gains momentum and can reach a point where it feels like a tractor-trailer rolling downhill with no brakes.
Wired for Intimacy is a timely and important book. Struthers offers keen and strategic insights from neurobiology and psychology. But what makes this book truly helpful is the fact that Struthers neither leaves his argument to neuroscience, nor does he use the category of addiction to mitigate the sinfulness of viewing pornography.
Sinners naturally look for fig leaves to hide sin, and biological causation is often cited as a means of avoiding moral responsibility. Struthers does not allow this, and his view of pornography is both biblical and theologically grounded. He lays responsibility for the sin of viewing pornography at the feet of those who willingly consume explicit images. He knows his audience—after all, his classrooms are filled with young male college students. The addict is responsible for his addiction.
At the same time, any understanding of how sin works its deceitful evil is a help to us, and understanding how pornography works in the male mind is a powerful knowledge. Pornography is a sin that robs God of his glory in the gift of sex and sexuality. We have long known that sin takes hostages. We now know another dimension of how this particular sin hijacks the male brain. Knowledge, as they say, is power.
Two Is Better Than One—Who Knew? [AlbertMohler.com » Blog] 2013-10-04 03:12
For some time now, scholars like W. Bradford Wilcox at the University of Virginia and Charles Murray of the American Enterprise Institute have been telling us that marriage is becoming an upper class phenomenon. More accurately, they have been pointing to the fact that lower-income Americans have been progressively abandoning marriage for the last two decades.
Now, along comes Derek Thompson, writing for The Atlantic, making many of the same points. Thompson points to an analysis of census data that reveals the vast economic consequences of this abandonment. Put bluntly, the failure to marry dramatically increases the likelihood of poverty and continued economic retreat.
According to this new data, the average American family with married parents and at least one child under age 18 living in the same home earned $81,000 last year. Interestingly, almost all of the actual growth in this average family’s income in recent years has come from the wife working. Thompson directs our attention to this fact in order to make his larger point: our marriage crisis is making income inequality worse. Those who are getting married and staying married are, on average, moving ahead in the economy. In contrast, those who are not married are falling behind—fast. Add to this the fact that when people marry, they tend to marry someone who shares the same work ethic. The strong get stronger and the weak get weaker.
As Thompson puts it: “In a strange twist, marriage has recently become a capstone for the privileged class. The decline of marriage, to the extent that we’re seeing it, is happening almost exclusively among the poor.”
Unrelated evidence for the importance of marriage comes from The Journal of Clinical Oncology. Researchers have documented the fact that on average married cancer patients live longer than unmarried patients. As Tara Parker-Pope of The New York Times explains, “Married cancer patients live longer than single people who have the disease, suggesting that logistical and emotional support from a loved one may be far more critical to cancer care than previously recognized.”
You will not be surprised to know that unmarried men are at greatest risk. Wives make a huge difference in the health habits of their husbands, right down to making sure that doctor’s appointments are made and medicines are taken. Nevertheless, married women also survive longer than unmarried women with the same disease. Even husbands really help. Single patients are far more vulnerable.
All this is testimony to the power of marriage, and to the fact that marriage is one of the greatest gifts God has given his human creatures.
Interestingly, Derek Thompson ends his article with these words: “This is the marriage crisis behind our inequality crisis. It is not complicated. It requires no regressions. It is the simplest math equation in the world. It says: Two is more than one.”
Really?
I think we know where that equation began: “Then the Lord God said, ‘It is not good that the man should be alone; I will make him a helper fit for him’” (Gen 2:18 ESV).
Or, as a modern paraphrase of that text might read: “Two is more than one.”
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
Derek Thompson, “How America’s Marriage Crisis Makes Income Inequality So Much Worse,” The Atlantic, Tuesday, October 1, 2013. http://www.theatlantic.com/business/archive/2013/10/how-americas-marriage-crisis-makes-income-inequality-so-much-worse/280056/
Tara Parker-Pope, “Marriage May Aid in Longevity,” The New York Times, Tuesday, October 1, 2013. http://well.blogs.nytimes.com/2013/09/24/married-cancer-patients-live-longer/?_r=0
Ayal A. Aizer, et. al., “Marital Status and Survival in Patients with Cancer,” The Journal of Clinical Oncology, JCO.2013.49.6489, Monday, September 23, 2013. http://jco.ascopubs.org/content/early/2013/09/18/JCO.2013.49.6489
The Indispensable Evangelical: Carl F. H. Henry and Evangelical Ambition in the Twentieth Century [AlbertMohler.com » Blog] 2013-10-04 02:18
I was very pleased to address the Carl F. H. Henry at 100: A Centennial Celebration conference at The Southern Baptist Theological Seminary last week. The event was both scholarly and deeply appreciative of Carl Henry and his legacy. In my address, “The Essential Evangelical: Carl F. H. Henry and Evangelical Ambition in the Twentieth Century,” I did my best to speak to Henry’s strategic role in the creation of the evangelical movement in America. I also sought to learn from Henry and his generation of evangelical leaders as we consider what ambitions evangelicals should serve today.
George and Barbara Witness a Wedding—When a Private Act Sends a Public Message [AlbertMohler.com » Blog] 2013-10-01 13:50
Former President George H. W. Bush and his wife Barbara attended a wedding a few days ago, and it made national news. According to The Washington Post, the elder Bushes attended the wedding of Bonnie Clement and Helen Thorgalsen, held at Kennebunkport, Maine. The two lesbians, co-owners of a general store in neighboring Kennebunk, were married in an outdoor celebration attended by family and friends. The 41st President of the United States was present, along with the former First Lady. Bonnie Clement told The Washington Post, “Who would be best to acknowledge the importance of our wedding as our friends and as the former leader of the free world? When they agreed to do so we just felt that it was the next acknowledgement of being ‘real and normal.’”
As it turns out, President Bush did not merely attend the wedding. He also served as an official witness, signing the legal documents for the ceremony and the Maine wedding license. Under a photograph with the former president the couple added the words, “Getting our marriage license witnessed!”
No one should be surprised by the opening line of the report in The Washington Post: “Another prominent Republican has come out in support of same-sex marriage—or, at least, in support of one particular same-sex marriage.” Similarly, the “Daily Intelligencer” column at New York Magazine declared that George and Barbara Bush are apparently in favor of same-sex marriage “since they not only attended a lesbian couple’s wedding on Saturday, but served as witnesses as well.”
The news coverage of the Bushes’ attendance at the same-sex wedding points to a reality that must be understood—and fast. Attendance at a wedding is not a neutral act. The history and context of the wedding ceremony identify all those present as agreeing to the rightness of the marriage and acting as witnesses to the exchange of vows. This is why the venerable language of The Book of Common Prayer, used in the overwhelming majority of Christian weddings, calls upon anyone with knowledge that the proposed union is invalid to speak, “or forever hold his peace.” Anyone remaining silent at that point is affirming the rightness and validity of the marriage, and all who are present are counted as both witnesses and those who celebrate the union.
This issue arose two years ago when controversy erupted over comments that Houston megachurch pastor Joel Osteen made on CNN’s Piers Morgan Tonight. In response to a question from Morgan, Osteen said that he would not officiate at a same-sex wedding. Morgan then pressed him by asking if Osteen would attend a same-sex wedding. Osteen replied:
Well, I haven’t been to many weddings lately to begin with and I’m talking about somebody that was, you know, dear to us. I’m not going to disrespect somebody that’s dear to us and say, you know what, you’re not good enough for us or something like that. That’s the way that I would see it. Now, I’m not going to just run off and go attend, you know, certain marriages just to make a statement because that’s not who I am and that’s not what I stand for and, again, I don’t look down on those people.
That is incoherence, and even Piers Morgan saw through it. It is incoherent to say that you cannot officiate at a same-sex wedding because you believe it to be wrong, and then turn around and say that you would attend a same-sex wedding and join in the celebration. Beyond incoherence, it is ministerial malpractice and bearing false witness.
We must certainly understand the relational challenges and the predicaments that this poses for Christians who do not believe that same-sex marriage is right in the sight of God. Those who would affirm same-sex marriage and the normalization of homosexuality must defy the clear teachings of Scripture. Christians cannot affirm what the Bible defines as sin, and yet that is what is demanded of us in our current cultural context. One of the hardest issues for every Christian will be the responsibility to relate to everyone we know with both love and truth.
But it is truth that protects love from dissolving into mere sentimentality. Likewise, it is love that prevents truth from being reduced to impersonal abstractions. At some point or another, almost all of us will be put into the situation Piers Morgan asked Joel Osteen to consider. At some point, we will either attend a same-sex ceremony, or we will not. Declining to attend will come with undeniable relational consequences, but so would attending. As one believer who struggles with same-sex attraction recently told me, “It does not help when fellow Christians send mixed signals.” We cannot allow our love to lapse into sentimentality, even as we love those who plan to enter into what we know is not and cannot be marriage. Note carefully that Bonnie Clement spoke of the Bushes’ presence at the wedding as a powerful affirmation that the union was “real and normal.”
A spokesman for President and Mrs. Bush said that the former first couple attended the wedding as “private citizens attending a private ceremony for two friends.” There are two problems with this account. First, if the Bushes were simply private citizens, there would have been no news story. After all, Bonnie Clement told the newspaper that President Bush had been invited as a friend “and as the former leader of the free world.” Needless to say, being identified as “former leader of the free world” is not a private matter. Second, a wedding is not actually a private affair. That marriage license was not filed with friends, but with a legal authority. And that legal document, available for public view as a public record, lists George H. W. Bush as an official witness to the union. The Washington Post had every good reason to declare that the former president had “come out in support of same-sex marriage.”
But, this is not just about the Bushes. The same predicament remains, even if we are not the former leader of the free world. To be present at a wedding is to affirm that it is right, whether you sign a legal document or not.
No one said this was going to be easy, and this is hardly the end of the predicaments and perplexities that will challenge Christians who stand on biblical teaching in the days ahead. This is one question, however, that Christians had better think through fast. A wedding invitation might soon be headed your way.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
The Reliable Source, “George H. W. Bush Is Witness at Same-Sex Marriage in Maine,” The Washington Post, Wednesday, September 25, 2013. http://www.washingtonpost.com/blogs/reliable-source/wp/2013/09/25/george-h-w-bush-is-witness-at-same-sex-marriage-in-maine/
Margaret Hartmann, “George H. W. Bush Served as Witness at a Lesbian Wedding,” New York Magazine, Wednesday, September 25, 2013. http://nymag.com/daily/intelligencer/2013/09/george-hw-bush-witness-at-lesbian-wedding.html
R. Albert Mohler, Jr., “Would You Attend a Same-Sex Wedding?,” Tuesday, October 18, 2011. http://www.albertmohler.com/2011/10/18/would-you-attend-a-same-sex-wedding/
R. Albert Mohler, Jr., “The Osteen Moment—Your Own Moment Will Come Soon Enough,” Thursday, January 27, 2011. http://www.albertmohler.com/2011/01/27/the-osteen-moment-your-own-moment-will-come-soon-enough/
The Cultural Revolution on the College Campus—Why it Matters to You [AlbertMohler.com » Blog] 2013-10-01 01:11
Several years ago, sociologist Peter Berger argued that secularization has been most pervasive in two social locations—Western Europe and the American college and university campus. The campuses of elite educational institutions are among the most thoroughly secularized places on our planet. This should concern anyone with an interest in higher education, of course. But it really matters to every American—or at least it should.
A wonderful and concise explanation of why this is so was provided in the pages of The Weekly Standard this week by David Gelernter, a professor of computer science at Yale University. In the course of making a proposal for the “reclamation” of higher education, Professor Gelernter wrote this very important paragraph:
Since the cultural revolution culminating in the 1970s, the left has run nearly all of the nation’s most influential, prestigious universities. Their alumni, in turn, run American culture—the broadcast networks, newspapers, the legal and many other professions, Hollywood, book publishing, and, most important, the massive, insensate, crush-everything-in-your-path mega-glacier known as the U.S. federal bureaucracy—and even more important than that, the education establishment charged with indoctrinating our children from kindergarten up.
That’s why it matters to you. And that’s how the future direction of the culture is set by the current culture of the elite colleges and universities. Many parents are unaware of how this happens. Their children may or may not attend one of the most prestigious colleges in the nation. But in almost any other institution they will study under professors who want to be associated with (or eventually hired by) one of those elite institutions. Exceptions to this pattern are rare, and the influence of these elite schools extends throughout the culture at large.
David Gelernter is in a position to know. After all, he is a professor at Yale. As he makes clear, what happens at Yale doesn’t stay at Yale.
David Genernter, “Back to School: A Reclamation Project for Higher Ed.,” The Weekly Standard, September 30, 2013. http://www.weeklystandard.com/articles/back-school_756489.html
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
Can Evangelical Chaplains Serve God and Country?—The Crisis Arrives [AlbertMohler.com » Blog] 2013-09-17 13:07
Can chaplains committed to historic biblical Christianity serve in the United States military? That question, though inconceivable to our nation’s founders, is now front and center. And the answer to that question will answer another, even more important question: Can religious liberty survive under America’s new moral order?
The repeal of the military’s “Don’t Ask, Don’t Tell” policy, coupled with the Supreme Court’s ruling that the Defense of Marriage Act is unconstitutional, set the stage for this crisis. The full normalization of same-sex relationships within the U.S. military is part of the unprecedented moral revolution that is now reshaping American culture at virtually every level.
The crisis in the chaplaincy arrived with these developments. The presenting issue is clear: Can a chaplain committed to historic biblical Christianity remain in military service? Does the normalization of homosexuality require that all members of the military, including chaplains, join the moral revolution, even if doing so requires them to abandon their biblical convictions?
The answer, at least from the advocates of the moral revolution, is that evangelical Christian chaplains must go—and Southern Baptist chaplains must go first.
In recent weeks, the North American Mission Board of the Southern Baptist Convention, the endorsing agency for SBC chaplains, formulated a set of policies on these issues. These policies are required of all SBC-endorsed chaplains, and the guidelines are clear. SBC chaplains are to minister in line with the biblical convictions of the SBC and its churches as made clear in our denomination’s confession of faith, The Baptist Faith & Message. Chaplains are to offer respect to all, respect for the religious liberty of all, and respect for the religious diversity represented within the armed forces. But evangelical chaplains cannot deny or compromise the Gospel of Jesus Christ. As the document states: ”Responsible pastoral care will seek to offer repentance and forgiveness, help and healing, and restoration through the mercy and grace of Jesus Christ’s sacrificial gift of love on the cross.”
At the same time, SBC endorsed chaplains—the largest single group of non-Catholic chaplains—cannot violate their own convictions by conducting or attending a same-sex marriage ceremony, and they certainly cannot bless such a union. They cannot minister in any context that “would give the appearance of accepting the homosexual lifestyle or sexual wrongdoing.”
In accordance with established U.S. military policy and law, all chaplains are free to minister in accordance with the teachings and beliefs of their own churches, even as they minister to all and respect the religious liberty of others. And yet, the great moral revolution of our times now threatens the continued service of chaplains committed to the moral teachings of historic Christianity.
That point was made abundantly clear in an article published on Monday, September 16, by Associated Baptist Press. The author of the article is Tom Carpenter, identified as co-chair of the Forum on the Military Chaplaincy and an elder in the Presbyterian Church (USA). Carpenter wastes no time in declaring his argument that Southern Baptist chaplains must immediately resign from military service. Given the guidelines set down by the Southern Baptist Convention’s endorsing agency, “the only honorable course is to resign from the military chaplaincy and return to civilian ministry.”
Carpenter insists:
The North American Mission Board has turned the Army motto on its head. They have forced their endorsed chaplains into the untenable position of either serving God or country. Given that choice, as men (NAMB forbids women to serve as ordained chaplains) of God the only honorable course of action for most will be to resign their commissions and return to civilian ministry.
Carpenter then asserts:
If these Southern Baptist chaplains were civilian pastors, there would be no problem. As civilians, they undisputedly have an absolute First Amendment right to believe, preach and counsel in accordance with their denominational tenets. But they are not civilians, and have a duty to not only God, but also country. It is instructive that they are not salaried by the NAMB but by the American taxpayer.
Yes, and they do not surrender their constitutional guarantee of religious liberty when they accept a commission as a military chaplain. Carpenter’s group was on the forefront of advocating for homosexual rights within the military, calling for “Don’t Ask, Don’t Tell” to be revoked and for the Defense of Marriage Act to be struck down. At the same time, his group assured the nation that this moral revolution would not lead to any major exodus of chaplains from the Armed Services. In fact, they accused evangelicals of “crying wolf” in warning of such a crisis. Now, Carpenter is openly calling for Southern Baptist chaplains to join the moral revolution or get out of the military.
Make no mistake, the moral revolution driven by those who demand the total normalization of homosexuality and same-sex relationships will not stop with the crisis over military chaplains. But at this moment, the chaplains are on the front lines of the great cultural and moral conflict of our times. This is a moment of crisis for the chaplains; but it is also a moment of crisis for the entire nation. If religious liberty is denied to evangelical Christian chaplains in the military, if they must surrender their convictions or their commissions, then religious liberty is lost in America, and the chaplains will be but the first casualties of this loss.
Southern Baptist chaplains have been singled out in this call for mass resignation, but they will not be alone. Thousands of Roman Catholic chaplains are committed by their church to the same moral convictions. Chaplains representing other evangelical churches and denominations will find themselves facing the same moment of decision. Muslim and Jewish chaplains who cannot endorse homosexuality and same-sex marriage will face the same challenge.
In reality, it is the entire nation that now faces this crisis. Is America ready to demand that military chaplains choose between serving God and serving their country? We will soon know the answer to that question.
We will also know the answer to another, even more urgent question: Where will every Christian church stand on this matter? The great theological divide between those churches and denominations committed to biblical Christianity and those who are given over to the spirit of the age has never been more clear. Indeed, the divide grows clearer day by day.
Also clear is this: Southern Baptist chaplains cannot surrender their commitment to Christ in order to maintain their commitment to ministry within the Armed Services. Furthermore, Southern Baptists will take their instruction from their own churches, not from those churches and denominations who are wearing out their knees bowing to Baal.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
Tom Carpenter, “SBC Chaplains Cannot Serve ‘God and Country,” Associated Baptist Press, Monday, September 16, 2013. http://www.abpnews.com/opinion/commentaries/item/8848-sbc-chaplains-cannot-serve-god-and-country#.Ujh8UGSDQXw
Mike Ebert, “NAMB Guidelines for Military Chaplains Updated to Address Same-Sex Unions,” Baptist Press, Friday, August 30, 2013. http://www.bpnews.net/BPnews.asp?ID=40991
Stronger Together, Serving Together, Sending Together—The State Baptist Conventions and the SBC [AlbertMohler.com » Blog] 2013-09-16 22:01
Fall brings the opening of the new school year, the energy of the season of autumn and, for Southern Baptists, the meeting of the state Baptist conventions. In coming weeks, most of our state conventions will be holding their annual meetings. Pastors and laypeople will gather from local churches and assemble as a convention of Baptist churches. There is a glory in these meetings, and they affirm our need for these state conventions and their ministries.
A younger generation of Southern Baptists may well be unaware of the importance of the state conventions and their work. They would be well-advised to attend their local state convention and catch a vision of what the Baptist churches in their states are doing together.
Americans are regularly reminded that states matter. Our political system respects the role of the individual states, and most Americans identify not only as citizens of the United States, but as residents of their respective states. This does not make our nation weaker. We are stronger because the states retain an important role in building communities and building the nations. As our national experience has shown, there is great gain in recognizing the priority of the local, even in the building of the nation.
In Southern Baptist life, the same is profoundly true of our state conventions. If the state conventions did not exist, we would have to invent them. There is a need for Baptist churches within every state to coordinate and combine their energies for the cause of the Great Commission and the task of reaching the communities in their own state and region. This does not weaken the Southern Baptist Convention—it makes us stronger.
Respect for the state conventions comes naturally to me. As a boy, I participated in camps and programs for children and young people. Soon after my conversion, I boarded a church bus and headed for Lake Yale, the assembly of the Florida Baptist Convention. The first real exposure I had to the scope and scale of Southern Baptist mission work came when I was a nine-year-old boy sitting in the auditorium at Lake Yale. I came back year after year, attending Royal Ambassador Camp and an assortment of camps and retreats and conferences. The imprint of those experiences remains on my life even now.
As a young man called to the ministry, I headed to Samford University where I received the gift of education for ministry from a school founded by Alabama Baptists—at least part of the tuition for my education came directly through the Alabama Baptist Convention. As a young ministerial student, I was exposed to preaching and evangelism through the Alabama state evangelism conferences and I saw the cooperative ministries work by attending the Alabama Baptist Convention annual meeting. When I was elected president of the student Ministerial Association, Samford’s president, Dr. Leslie S. Wright, invited me to attend the Alabama Baptist State Board of Missions with him. I learned how Baptists work together.
Later, as a pastor and seminary student, I saw the cooperative ministries of the Kentucky Baptist Convention and was able to participate in its work. Later, I was elected editor of The Christian Index and shifted my ministry to the context of the Georgia Baptist Convention. I was immersed in the life of that state convention, and I saw first-hand that it was doing important work that would otherwise be left undone.
When disaster strikes, state disaster relief teams are first on the scene. When a pastor needs help, the state convention is close at hand. When strategies for reaching America’s urban areas are developed, state conventions are on the front lines. State conventions remember the rural churches and are there to combine strengths and walk alongside those congregations serving the heartland.
At the same time, the state conventions have the world on their hearts. Increasingly, our leading state conventions are increasing their commitment to the support of national ministries and the reaching of the nations. Many of these conventions have taken courageous steps to send a greater percentage of Cooperative Program funds to the cause of reaching the nations with the Gospel of Jesus Christ. These state conventions have made sacrifices for the Great Commission cause and are mobilizing churches to reach not only their communities, but the world.
Now is the time for Southern Baptists committed to the Great Commission to show up and support our state conventions, to attend our annual convention meetings, and to support every effort to reach our individual states, our nation, and the nations with the Gospel.
As a committed Southern Baptist, I would not know who I am without the state conventions that have contributed so much to my life and ministry. As president of The Southern Baptist Theological Seminary, I am proud and thankful to be in partnership with every one of our state conventions, and I want my students and faculty to share this pride and gratitude.
So, as the Southern Baptists in your state head for their annual meetings, determine to join them, to pray for them, to support them in Cooperative Program giving, and to strengthen the Great Commission vision and energy you will find there. Southern Baptists will never be bolder in mission and ministry than when we strengthen these bonds and stand together. Bring the full wealth of your conviction and the full passion of your desire for reaching your state, our nation, and all nations with the Gospel of Jesus Christ. Stronger together. Serving together. Sending together.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
The Man from Issachar—An Address at the Inauguration of Russell D. Moore [AlbertMohler.com » Blog] 2013-09-12 02:21
THE MAN FROM ISSACHAR
An Address Delivered in the City of Washington, D.C. upon the Inauguration of Russell D. Moore as President of the Ethics and Religious Liberty Commission of the Southern Baptist Convention on Tuesday, September 10, 2013 at Capitol Hill Baptist Church by R. Albert Mohler, Jr., President of The Southern Baptist Theological Seminary
Without a providential understanding of time and history, one is left with the affirmation that human affairs are often guided by a series of very happy coincidences. At just the right time, the right leader emerges to fill a crucial need. The intersection of an individual life and a demonstrable need meet in a moment and in a person. We celebrate just such an intersection today, but I am not able to describe it as a coincidence. I believe that the providence of God is today demonstrated in the intersection of a man and a moment—in the inauguration of Russell D. Moore as President of the Ethics and Religious Liberty Commission of the Southern Baptist Convention.
First, I point to the character and giftedness of this man. I can remember the very first conversation I had with Russell Moore. In that first meeting, I caught a glimpse of his intelligence, his conviction, and his ambitions. I knew then that he was out to change the world, but that his first loyalty and constant horizon is not this world, but the world that is already but not yet—in other words, not the kingdoms of this world but the Kingdom of our God and of His Christ.
His intellect is first rate, as is his scholarship. He came as a Doctor of Philosophy student and transformed his doctoral dissertation into a manifesto for kingdom ministry and cultural engagement. His intelligence is energetic and his wit always on hand. To talk with Russ is to enter into a world of ideas undergirded by conviction and footnoted with readings.
He is not merely fascinated by ideas, he is a true public intellectual. He belongs to that class of thinkers who are not merely collectors of ideas but movers of minds. He is a master of communicating those ideas and he knows how to make truth come alive as a living force.
He is one of the most natural conversationalists I have ever encountered. He is like the Victorians who could enter any room and join the conversation and immediately add to it. He is a voracious reader who is a walking bibliography and a library on legs. He comes alive when a book or an idea or a problem or a personality comes to attention.
Amitai Etzioni has distinguished between two classes of public intellectuals: those who are generalists (who can speak about anything intelligently) and those who are disciplinary (who can speak with unique authority within a specific field). Russ combines the best of both. He can talk about almost anything; but he talks with the authority of one who knows of what he speaks.
Above all, Russell Moore is a Christian thinker. In this construction, “Christian” operates as a noun, not as an adjective. He does not merely think like a Christian, he thinks as a Christian. His personal commitment to Christ, to the total truthfulness and trustworthiness of the Word of God, and to the faith once for all delivered to the saints is clear and tested. He is a defender of the faith and a Christian intellectual who dearly and deeply loves the Christian faith.
Russell Moore is a Baptist by conviction and a Southern Baptist by passion. He is a member of the tribe who transcends tribalism. He is not a Baptist by accident. His commitment to the free church in a free state and to the elegant simplicity of Baptist ecclesiology is clear. He is a conversionist and a churchman. He is deeply committed to the Gospel of Jesus Christ and to the Great Commission. He knows the Southern Baptist Convention and he loves Southern Baptists with an eyes-open love. Thus, he can lead Southern Baptists. The late Carlyle Marney once said of Southern Baptists, “We may not be much but we are many.” Russ Moore is representative of a generation of leaders needed to make much of many.
He is, as no less than Augustine described the Christian teacher, one who is passionately committed to truth because he stakes his life on this truth and is himself transformed by this truth. He is, as our common mentor Carl F. H. Henry would define, a Christian thinker who is unreservedly committed to the totality of the comprehensive truth claim of the Christian world and life view.
All that, and he has a sense of humor. Russ Moore has an ear for irony and a readiness to be found joyful. Like G. K. Chesterton and C. S. Lewis, he knows that the deepest truths reveal the deepest joys, even as the reality of our human foibles reveals humor, whether we like it or not. Like Flannery O’Connor, he has an eye for the bare reality of truth, knowing, as Flannery would remind us, “Truth does not change according to our ability to stomach it.”
He is a leader who knows how to run a great enterprise. At a very early age he became Dean of the School of Theology at the mother seminary of the Southern Baptist Convention, serving also as its Senior Vice President for Academic Administration. His reputation as a leader is well attested. He is a leader, an administrator, and an energetic catalyst for good. At the Ethics and Religious Liberty Commission he comes to a work well established and much respected. He will build it and take it into the future.
He is also a faithful husband and a compassionate father. To know Russ is to know that he is the husband of Maria, and the father of Benjamin, Timothy, Samuel, Jonah, and Taylor. He finds joy in his home, and he has a joyful home in which to establish his life, both public and private. His dependence upon Maria is transparent, as is his joy in his sons.
He is a theologian of conviction, a leader of great ability, a teacher of righteousness, a preacher of rare ability and power, and a thinker who knows how and when and where to think out loud. He is an ethicist by reflex, by training, and by experience. He is a colleague with whom I have spent countless hours in joyful conversation and gone through times of trial and great challenge. I know what he is made of. I know where he comes from. I know who he is. I know his ambitions. He is not a self-made man, but a man well made for these times.
So we know the man, but what of the times? Twenty-five years ago, Carl Henry warned:
Our generation is lost to the truth of God, to the reality of divine revelation, to the content of God’s will, to the power of his redemption, and to the authority of His Word. For this loss it is paying dearly in a swift relapse to paganism. The savages are stirring again; you can hear them rumbling and rustling in the tempo of our times.[1]
The last quarter century since Henry’s statement of our crisis has brought no reversal of the trends he observed. To the contrary, the formerly Christian West is, in many sectors, so thoroughly secularized that it now has no consciousness of even being so. The Christian truth claim was reduced to a Christian memory, and now even that memory is gone. Our confidence in American exceptionalism is now fully shaken. If anything, America now seems to be secularizing in a delayed pattern, as compared to Europe, but perhaps even faster on its present course. As the Canadian philosopher Charles Taylor reminds us, for millions of people in our civilization, and especially among the elites, belief in God is now, according to their own thinking, virtually impossible.
Many decades ago, the Quaker philosopher Elton Trueblood identified America as a “cut-flower civilization”—its flower cut off from the only source of its sustenance. Those roots have further receded from the cultural horizon.
We are now in the midst of a moral revolution marked by a comprehensive scope and velocity that are perhaps without precedent in human experience. We find ourselves looking at a moral world that is changing right before our eyes, and many Christians seem both bewildered and fearful—precisely because they are.
But the real crisis is not in the world, but in the church. More than sixty years ago, Carl Henry (whose 100th birthday we would mark this year), reminded the evangelicals of that day that the failure was ours before it was a failure in the world.
It was the failure of Fundamentalism to work out a positive message within its own framework, and its tendency instead to take further refuge in a despairing view of world history, that cut off the pertinence of evangelicalism to the modern global crisis. The really creative thought, even if done in a non-redemptive context, was now being done by non-evangelical spokesmen.[2]
Through this analysis of the problem, what Henry called The Uneasy Conscience of Modern Fundamentalism, he called evangelicals to a new mode of cultural and intellectual engagement.
“There is a rising tide of reaction in Fundamentalism today—a reaction born of uneasy conscience and determined no longer to becloud the challenge of the Gospel in modern times,” Henry wrote. “It is a reaction to which the best minds of evangelicalism are bending their effort these days, convinced that no synthesis is more relevant than modern frustration and biblical redemptionism.”[3]
In other words, he saw a generation coming, and he saw the likes of Russell Moore on the horizon. We dare not underestimate the challenges before us. We are living in a cut-flower civilization. There is a new paganism growing rapidly around us. There are threats to human life and human flourishing at every hand. We do see the ramparts of the family and the faith being both scaled and taken down. Religious liberty is under direct threat and we find ourselves in a moment of great civilizational peril. The culture of death is now institutionalized and made more ominous yet by technology. America has grown more polarized within and seems to be without a clear sense of itself within the international order. The most fundamental, essential, and pre-political institutions of human life and culture are now up for radical revision to the point of destruction. The scale of the crisis defies exaggeration.
And yet, these are precisely the conditions for optimal Christian witness. Under these conditions, the keenest edge of Christian thinking is soon evident and the operation of a genuinely Christian mind is transformative. The church is revealed to be what we know it to be, the kingdom community of the blood-bought, deployed in this world even as we belong truly to the world to come. This is no time for the weak-kneed or for weak thinking. These times call forth the deepest level and highest quality of Christian thinking, cultural engagement, Gospel-mindedness, strategic ambition, and churchly demonstration.
We do not choose our times, but this is a time for choosing. In the last era of the Roman Empire, Bishop Augustine chose to find his bearings for the City of Man within the greater love of the City of God. A time of crisis can bring us to surrender and lose heart, or it can produce The City of God or the Letter from Birmingham Jail.
I think Russ Moore’s legendary love of country music will serve him well. He knows how to speak of brokenness answered with hope and mercy. And he knows, as Johnny Cash would remind us, “there’s a man goin’ round, taking names.”
In 1 Chronicles 12:32, we read of the men of the tribe of Issachar, “who had understanding of the times, to know what Israel ought to do.” We know Russell Moore as a man from Mississippi. I think he is really a man from Issachar. I think he has an understanding of the times, and he knows what God’s people ought to do.
The man and the moment have come together and, like you, I don’t for a moment believe it is a coincidence.
[1] Carl F. H. Henry, Twilight of a Great Civilization: The Drift Toward Neo-Paganism (Westchester, Illinois: Crossway Books, 1988), 15.
[2] Carl F. H. Henry, The Uneasy Conscience of Modern Fundamentalism (Grand Rapids: Eerdmans, 1947), 32.
[3] Ibid, 34.
Preaching with Authority: Three Characteristics of Expository Preaching [AlbertMohler.com » Blog] 2013-09-06 02:12
Authentic expository preaching is marked by three distinct characteristics: authority, reverence, and centrality. Expository preaching is authoritative because it stands upon the very authority of the Bible as the word of God. Such preaching requires and reinforces a sense of reverent expectation on the part of God’s people. Finally, expository preaching demands the central place in Christian worship and is respected as the event through which the living God speaks to his people.
A keen analysis of our contemporary age comes from sociologist Richard Sennett of New York University. Sennett notes that in times past a major anxiety of most persons was loss of governing authority. Now, the tables have been turned, and modern persons are anxious about any authority over them: “We have come to fear the influence of authority as a threat to our liberties, in the family and in society at large.” If previous generations feared the absence of authority, today we see “a fear of authority when it exists.”
Some homileticians suggest that preachers should simply embrace this new worldview and surrender any claim to an authoritative message. Those who have lost confidence in the authority of the Bible as the word of God are left with little to say and no authority for their message. Fred Craddock, among the most influential figures in recent homiletic thought, famously describes today’s preacher “as one without authority.” His portrait of the preacher’s predicament is haunting: “The old thunderbolts rust in the attic while the minister tries to lead his people through the morass of relativities and proximate possibilities.” “No longer can the preacher presuppose the general recognition of his authority as a clergyman, or the authority of his institution, or the authority of Scripture,” Craddock argues. Summarizing the predicament of the postmodern preacher, he relates that the preacher “seriously asks himself whether he should continue to serve up monologue in a dialogical world.”
The obvious question to pose to Craddock’s analysis is this: If we have no authoritative message, why preach? Without authority, the preacher and the congregation are involved in a massive waste of precious time. The very idea that preaching can be transformed into a dialogue between the pulpit and the pew indicates the confusion of our era.
Contrasted to this is the note of authority found in all true expository preaching. As Martyn Lloyd-Jones notes:
Any study of church history, and particularly any study of the great periods of revival or reawakening, demonstrates above everything else just this one fact: that the Christian Church during all such periods has spoken with authority. The great characteristic of all revivals has been the authority of the preacher. There seemed to be something new, extra, and irresistible in what he declared on behalf of God.
The preacher dares to speak on behalf of God. He stands in the pulpit as a steward “of the mysteries of God” (1 Cor 4:1) and declares the truth of God’s word, proclaims the power of that word, and applies the word to life. This is an admittedly audacious act. No one should even contemplate such an endeavor without absolute confidence in a divine call to preach and in the unblemished authority of the Scriptures.
In the final analysis, the ultimate authority for preaching is the authority of the Bible as the word of God. Without this authority, the preacher stands naked and silent before the congregation and the watching world. If the Bible is not the word of God, the preacher is involved in an act of self-delusion or professional pretension.
Standing on the authority of Scripture, the preacher declares a truth received, not a message invented. The teaching office is not an advisory role based on religious expertise, but a prophetic function whereby God speaks to his people.
Authentic expository preaching is also marked by reverence. The congregation that gathered before Ezra and the other preachers demonstrated a love and reverence for the word of God (Neh 8). When the book was read, the people stood up. This act of standing reveals the heart of the people and their sense of expectation as the word was read and preached.
Expository preaching requires an attitude of reverence on the part of the congregation. Preaching is not a dialogue, but it does involve at least two parties—the preacher and the congregation. The congregation’s role in the preaching event is to hear, receive, and obey the word of God. In so doing, the church demonstrates reverence for the preaching and teaching of the Bible and understands that the sermon brings the word of Christ near to the congregation. This is true worship.
Lacking reverence for the word of God, many congregations are caught in a frantic quest for significance in worship. Christians leave worship services asking each other, “Did you get anything out of that?” Churches produce surveys to measure expectations for worship: Would you like more music? What kind? How about drama? Is our preacher sufficiently creative?
Expository preaching demands a very different set of questions. Will I obey the word of God? How must my thinking be realigned by Scripture? How must I change my behavior to be fully obedient to the word? These questions reveal submission to the authority of God and reverence for the Bible as his word.
Likewise, the preacher must demonstrate his own reverence for God’s word by dealing truthfully and responsibly with the text. He must not be flippant or casual, much less dismissive or disrespectful. Of this we can be certain, no congregation will revere the Bible more than the preacher does.
If expository preaching is authoritative, and if it demands reverence, it must also be at the center of Christian worship. Worship properly directed to the honor and glory of God will find its center in the reading and preaching of the word of God. Expository preaching cannot be assigned a supporting role in the act of worship—it must be central.
In the course of the Reformation, Luther’s driving purpose was to restore preaching to its proper place in Christian worship. Referring to the incident between Mary and Martha in Luke 10, Luther reminded his congregation and students that Jesus Christ declared that “only one thing is necessary,” the preaching of the word (Luke 10:42). Therefore, Luther’s central concern was to reform worship in the churches by re-establishing there the centrality of the reading and preaching of the word.
That same reformation is needed in American evangelicalism today. Expository preaching must once again be central to the life of the church and central to Christian worship. In the end, the church will not be judged by its Lord for the quality of its music but for the faithfulness of its preaching.
When today’s evangelicals speak casually of the distinction between worship and preaching (meaning that the church will enjoy an offering of music before adding on a bit of preaching), they betray their misunderstanding of both worship and the act of preaching. Worship is not something we do before we settle down for the word of God; it is the act through which the people of God direct all their attentiveness to the one true and living God who speaks to them and receives their praises. God is most beautifully praised when his people hear his word, love his word, and obey his word.
As in the Reformation, the most important corrective to our corruption of worship (and defense against the consumerist demands of the day) is to rightly return expository preaching and the public reading of God’s word to primacy and centrality in worship. Only then will the “missing jewel” be truly rediscovered.
I am always glad to hear from readers. Write me at mail@albertmohler.com. Follow regular updates on Twitter at www.twitter.com/AlbertMohler.
Previously:
Expository Preaching: The Antidote to Anemic Worship
The Sheer Weightlessness of So Many Sermons: Why Expository Preaching Matters
The Expositors Summit 2013 at Southern Seminary—October 29-31
The goal of Christian preaching is nothing less than the glory of God in the Christlikeness of his people. For this reason, The Expositors Summit 2013 at The Southern Baptist Theological Seminary will seek to restore the primacy of expository preaching to the pulpits of local churches. Pastors, students, and all who love the Scriptures are invited to come hear Dr. R. Albert Mohler, Jr., H.B. Charles Jr., and Alistair Begg as the keynote speakers (along with other gifted leaders in various seminars).
Is Everything Bigger and Better in Texas? [Desiring God Blog] 2013-10-31 13:52

What is so good about the gospel? Have you ever asked yourself that question? What's the biggest and best thing God promises us? What is it exactly that makes it such good news?
In a sermon last week in Houston, Piper unwrapped “The Sweetest Good of the Good News”. And it's something bigger and better than forgiveness, freedom, or an eternity of your favorite hobby — shopping, golfing, or watching movies. Yes, even bigger and better than Houston, San Antonio, and Big D. The greatest good of the gospel is God himself.
It was one of several new messages from Piper over the last week. Below is the buffet of conference talks and sermons from recent trips to Idaho and Texas. You will find something for the Northerner and Southerner alike, for the proud potato harvester, the rough longhorn wrangler, and everyone in between.
How can God truly love us when he demands that we worship him? Does his desire for his own glory deny his devotion to us? As only he can, Pastor John shows us again that our greatest satisfaction will only ever be found in his glory.
“Why Is the Glory of God at Stake in Making People Plural?”
If the word 'peoples,' sound like a typo to you, you really need to hear this missions message. Piper draws out the implications of this one little word and shows how the Bible shapes the Great Commission with its language. The task is clear and unfinished, and we need to engage the places — the peoples — yet unreached with the gospel.
“A Spectacular and Scary Promise”
Paul promises in Romans 8 that those who are God's children are also his heirs — heirs of an eternal glory that will eventually be revealed to us. It is a promise so spectacular we can’t even imagine how good it is. But this promise comes to us through suffering — really, really scary pain, disease, conflict, failure, etc.. Amazingly, when we finally receive our inheritance, all our suffering will look as nothing by comparison.
“Saying Well As a Way of Seeing Wonder”
Reflecting on the life and poetry of George Herbert, Pastor John pled with pastors to write. He argues that the act of finding words to describe the worth of Christ is a way of seeing further and deeper into who he is.
The Reformation: Trick or Treat? [Desiring God Blog] 2013-10-31 01:25

It’s no accident that October 31 is both Halloween and the day remembered for the start of the Reformation. Both key off November 1, All Saints’ Day — or All Hallows’ Day (Hallows from the Latin for saints or holy ones).
On All Hallows’ Eve, October 31, 1517, the Roman Church received the world’s most memorable trick-or-treater at its door — though barely noticed at the time — when a lowly priest named Martin Luther approached the threshold of the Wittenberg branch in Germany and posted his 95 measly theses (they aren’t nearly as impressive as you would expect). The coming All Saints’ Day seemed like an excuse for sparring about the Church’s deplorable sanctioning of indulgences, and Luther was angling for some good-spirited debate.
But the Church was centuries overdue for major reform, the kindling was in place, and Luther’s little, almost accidental spark set the whole thing ablaze. Some nameless visionary translated his theses from the Church’s Latin into the people’s German and sent them far and wide through the printing press. In time, this lowly monk proved to have what it took to hold his ground against the Church and the world — “Here I stand,” he said courageously before the emperor — and under God, he became the human tip of the spear for massive reform.
Of course, that’s the reductionistic version of the story. Save his own Son, God doesn’t change the world through a single person, but through people. With and behind every remembered individual is some great collective. Luther had a significant supporting cast in his Wittenberg work, and on the grander scale, it took many others — like Ulrich Zwingli, John Calvin, Martin Bucer, Thomas Cranmer, John Knox, and many more, all with their associates and assistants — to usher in reform far and wide. God gave Luther the bullhead to do the pioneering. He was the battering ram. But five centuries of Protestant Christianity wouldn’t have followed in the wake of Luther alone.
In particular, Calvin’s thinking, writing, and systematizing played a complementary role to Luther’s pioneering flair. Born in 1509 in France, Calvin was only eight years old when Luther played his Halloween trick in 1517.
Calvin was trained as a humanist and converted sometime between 1528 and 1532, while at university, and by All Saints’ Day, 1533, he had himself in hot water. Sixteen years after Luther posted his theses, Calvin’s friend Nicolas Cop delivered an All Saints’ convocation heralding Christ as the sole mediator (not the “saints”). Some suspected this patently Protestant address was written by Calvin, and he soon found himself on the run.
As an exile, Calvin spent time in Basel, and seemingly by accident came to Geneva for a single night in 1536 on his way to Strasbourg for an ivory-tower, academic life of study and writing. The fiery Swiss reformer William Farel learned Calvin was in town and prevailed upon him to join the reformation cause in Geneva. Calvin acquiesced, and stayed there in Geneva — minus a three-year exile from 1538–1541 — until his death in 1564 at age 54.
Reformation Day is ripe for remembering an array of biblical truths — that the Scriptures are our only final authority (sola Scriptura); that God accepts us by grace alone, through faith alone, on the basis of Christ alone (justification); that God often uses the unlikeliest of people to turn the world upside down; that God doesn’t just raise up great individuals, but collections of people, veritable teams, each with his lot, and his own local cohort, to bring about widespread change; and all these conspiring to the glory of God alone (soli Deo gloria).
But here’s one to keep on your radar this year. God loves to use the seeming accidents in our lives to bring about his purposes. It’s the “accidents” that remind us we’re emphatically not the captain of our own soul, we’re not piloting our own destinies, we’re not on the block for planning the whole thing out and executing on it. How sad a course it would be if we cooked up the whole thing out as we came of age and spent the rest of our lives living out our boring and uncreative little visions?
That such a Reformation began almost 500 years ago, and continues to this day — this is your story too — is not the result of any human plan. It has been the “accidents” which have given it the markings of divine fingerprints — Luther’s accidental spark that first lit the flame and Calvin’s accidental lone night in Geneva that changed the course for that city and for a major branch of Protestant theology.
Reformation Day is a reminder to embrace the “accidents” in our lives, look for the hand of providence, and trust that his plans for us are better than our wildest dreams. For those who are his, he truly works together for their good all things — even and especially the seeming accidental — to do for us far more abundantly than all that we ask or think (Romans 8:28; Ephesians 3:20).
More for Reformation Day from Desiring God:
eBook: Martin Luther: Lessons from His Life and Labor (free PDF, mobi, and epub)
Book: John Calvin and His Passion for the Majesty of God (free PDF)
eBook: Portrait of Calvin (free PDF, mobi, and epub)
Book: With Calvin in the Theater of God: The Glory of Christ and Everyday Life (free PDF)
Article: When Jesus Haunts Your Halloween
Article: Trick or Treat: It’s Martin Luther
Video: After Darkness, Light: John Piper on John Calvin (on Location in Geneva) —
Jesus Saves [Desiring God Blog] 2013-10-30 01:00

The Son of Man came to seek and to save the lost. (Luke 19:10)
Christianity is summed up in these words: Jesus came to seek and save the lost. If we were asked to describe in a sentence the heart of the gospel, there it is.
There is no other news like this. Every other religion says it backwards. Every other religion tells us to seek. We are advised to climb trees like Zacchaeus, to depend upon our own exertion for any hope of ascending to the divine. We are told to bridge the gulf by our effort. If you want salvation, they say, then seek it.
In a sense, that is the world — we live in a planet full of seekers. We are, in one way or another, tree-climbers, maneuvering ourselves to gain some advantage, to achieve some perspective, to find personal peace. And then Jesus comes.
We are lost in our own seeking until Jesus comes and says to us, “Hurry and come down” (Luke 19:5). Stop your searching. Stop trying to save yourself. I have come to seek and save the lost.
Our exertion is then silenced. All our seeking — our trying to reach the divine on our own — is silenced when we learn that the divine has reached down to us . . . by becoming one of us. Here we are, spinning our wheels in hopes of getting God, and then God, despite our belittling works, comes to get us. That gulf we couldn’t bridge is the burden he takes upon himself.
We were lost, sinners who rightly deserve God’s judgment. And Jesus came to take the judgment for us. He suffered in our place on the cross, was dead and buried, and then on the third day was raised to life. He ascended to the Father’s right hand from where he reigns over all. Jesus sought us, and he has saved us, if we trust him. Do you believe this? Do you feel the wonder of this salvation?
Jesus, you are the one who saves, not us. Thank you for rest, for hushing the furious winds of our faithless works. Thank you for stopping the strivings of our souls. Overcome us more and more with the glory of your grace, and make our posture toward others echo this summary of your gospel: “the Son of Man came to seek and to save the lost.”
Recent posts from Jonathan Parnell:
God’s Glory Wins [Desiring God Blog] 2013-10-29 11:41

Not everyone will believe the gospel. Why?
“God desires all people to be saved,” 1 Timothy 2:4 tells us. “God does not take pleasure in the death of anyone,” Ezekiel 18:32 says. Then why are there some who refuse to trust in Jesus and therefore die lost in their sins?
There are two different answers to this question.
But we should understand that these two answers go beyond making sense of God’s will of decree and will of command. Those “two wills” in God describe a biblical distinction that’s been expressed various ways in the Scriptures and throughout the centuries. God’s “two ways of willing,” writes John Piper, “implies that God decrees one state of affair while also willing and teaching that another state of affairs should come to pass” (Does God Desire All to Be Saved?, 16). This means that though God desires all people to be saved (his “will of command”), only those chosen in Christ will believe the gospel and be saved (his “will of decree”). But true as it is, this explanation still falls short of getting to the why. Why is this the case? Why does God not decree all that he prescribes?
And here is where we face those two different answers.
One answer is that there is something more powerful than God that is able to frustrate his will. It says that God is nice to desire all people to be saved, but he doesn’t have the strength to make it happen. The second answer says, in Piper’s words, “God wills not to save all, even though he ‘desires’ that all be saved, because there is something else that he wills or desires more, which would be lost if he exerted his sovereign power to save all” (emphasis added, 39).
The second answer is one that both Calvinists and Arminians can affirm. Both say that God doesn’t save everyone because he is committed to something more than saving everyone. The difference between Calvinists and Arminians is seen in what that higher commitment is.
Piper explains,
The answer the Arminians give is that human self-determination and the possible resulting love relationship with God are more valuable than saving all people by sovereign, efficacious grace. The answer the Reformed give is that the greater value is the manifestation of the full range of God’s glory in wrath and mercy (Romans 9:22–23) and the humbling of man so that he enjoys giving all credit to God for his salvation (1 Corinthians 1:29). (39)
So one explanation says that the higher commitment is God leaving the destiny of our eternal souls up to our own decision-making. The higher commitment is God securing our right to let our choices be the decisive factor in where we spend eternity. The other explanation — the Calvinist answer — says that God’s higher commitment is the full display of his glory. God’s glory wins, which means that his just wrath is poured out on all unrighteousness, and his mercy is lavished on all whom he loves.
God’s highest commitment — beyond his moral will that all people everywhere repent —is that the full panorama of his glory shine forth. That glory is his mercy, grace, steadfast love and faithfulness, and his refusal to by no means clear the guilty . . . so that the vessels of his mercy might know the riches of his glory (Exodus 34:6–7; Romans 9:23).
Related resources:
Does God Desire All to Be Saved? (Crossway, 2013)
Parents, Require Obedience of Your Children [Desiring God Blog] 2013-10-29 01:00

I am writing this to plead with Christian parents to require obedience of their children. I am moved to write this by watching young children pay no attention to their parents’ requests, with no consequences. Parents tell a child two or three times to sit or stop and come or go, and after the third disobedience, they laughingly bribe the child. This may or may not get the behavior desired.
Last week, I saw two things that prompted this article. One was the killing of 13-year-old Andy Lopez in Santa Rosa, California, by police who thought he was about to shoot them with an assault rifle. It was a toy gun. What made this relevant was that the police said they told the boy two times to drop the gun. Instead he turned it on them. They fired.
I do not know the details of that situation or if Andy even heard the commands. So I can’t say for sure he was insubordinate. So my point here is not about young Lopez himself. It’s about a “what if.” What if he heard the police, and simply defied what they said? If that is true, it cost him his life. Such would be the price of disobeying proper authority.
I witnessed such a scenario in the making on a plane last week. I watched a mother preparing her son to be shot.
I was sitting behind her and her son, who may have been seven years old. He was playing on his digital tablet. The flight attendant announced that all electronic devices should be turned off for take off. He didn’t turn it off. The mother didn’t require it. As the flight attendant walked by, she said he needed to turn it off and kept moving. He didn’t do it. The mother didn’t require it.
One last time, the flight attendant stood over them and said that the boy would need to give the device to his mother. He turned it off. When the flight attendant took her seat, the boy turned his device back on, and kept it on through the take off. The mother did nothing. I thought to myself, she is training him to be shot by police.
The defiance and laziness of unbelieving parents I can understand. I have biblical categories of the behavior of the spiritually blind. But the neglect of Christian parents perplexes me. What is behind the failure to require and receive obedience? I’m not sure. But it may be that these nine observations will help rescue some parents from the folly of laissez-faire parenting.
“Children, obey your parents in the Lord, for this is right” (Ephesians 6:1). It makes no sense that God would require children to obey parents and yet not require parents to require obedience from the children. It is part of our job — to teach children the glory of a happy, submissive spirit to authorities that God has put in place. Parents represent God to small children, and it is deadly to train children to ignore the commands of God.
Obedience is not merely a “legal” category. It is a gospel category. Paul said that his gospel aim was “to bring about the obedience of faith” (Romans 1:5). He said, “I will not venture to speak of anything except what Christ has accomplished through me to bring the Gentiles to obedience — by word and deed” (Romans 15:18).
Paul’s aim was “to take every thought captive to obey Christ” (2 Corinthians 10:5). He required it of the churches: “If anyone does not obey what we say in this letter, take note of that person, and have nothing to do with him” (2 Thessalonians 3:14).
Parents who do not teach their children to obey God’s appointed authorities prepare them for a life out of step with God’s word — a life out of step with the very gospel they desire to emphasize.
(If anyone doubts how crucial this doctrine is, please consider reading Wayne Grudem’s chapter, “Pleasing God by Our Obedience: A Neglected New Testament Teaching” in For the Fame of God’s Name, edited by Justin Taylor and Sam Storms.)
To watch parents act as if they are helpless in the presence of disobedient children is pitiful. God requires that children obey because it is possible for parents to require obedience. Little children, under a year old, can be shown effectively what they may not touch, bite, pull, poke, spit out, or shriek about. You are bigger than they are. Use your size to save them for joy, not sentence them to selfishness.
One explanation why children are out of control in public is that they have not been taught to obey at home. One reason for this is that many things at home don’t seem worth the battle. It’s easier to do it ourselves than to take the time and effort to deal with a child’s unwillingness to do it. But this simply trains children that obedience anywhere is optional. Consistency in requiring obedience at home will help your children be enjoyable in public.
If you tell a child to stay in bed and he gets up anyway, it is simply easier to say, go back to bed, than to get up and deal with the disobedience. Parents are tired. I sympathize. For more than 40 years, I’ve had children under eighteen. Requiring obedience takes energy, both physically and emotionally. It is easier simply to let the children have their way.
The result? Uncontrollable children when it matters. They have learned how to work the angles. Mommy is powerless, and daddy is a patsy. They can read when you are about to explode. So they defy your words just short of that. This bears sour fruit for everyone. But the work it takes to be immediately consistent with every disobedience bears sweet fruit for parents, children, and others.
One reason parents don’t require discipline is they have never seen it done. They come from homes that had two modes: passivity and anger. They know they don’t want to parent in anger. The only alternative they know is passivity. There is good news: this can change. Parents can learn from the Bible and from wise people what is possible, what is commanded, what is wise, and how to do it in a spirit that is patient, firm, loving, and grounded in the gospel.
Children need to obey before they can process obedience through faith. When faith comes, the obedience which they have learned from fear and reward and respect will become the natural expression of faith. Not to require obedience before faith is folly. It’s not loving in the long run. It cuts deep furrows of disobedient habits that faith must then not infuse, but overcome.
Laissez-faire parenting does not produce gracious, humble children. It produces brats. They are neither fun to be around, nor happy themselves. They are demanding and insolent. Their “freedom” is not a blessing to them or others. They are free the way a boat without a rudder is free. They are the victims of their whims. Sooner or later, these whims will be crossed. That spells misery. Or, even a deadly encounter with the police.
Since parents represent God to children — especially before they can know God through faith in the gospel — we show them both justice and mercy. Not every disobedience is punished. Some are noted, reproved, and passed over. There is no precise manual for this mixture. Children should learn from our parenting that the God of the gospel is a consuming fire (Hebrews 12:7, 29) and that he is patient and slow to anger (1 Timothy 1:16). In both cases — discipline and patience — the aim is quick, happy, thorough obedience. That’s what knowing God in Christ produces.
Parents, you can do this. It is a hard season. I’ve spent more than sixty percent of my life in it. But there is divine grace for this, and you will be richly rewarded.
More from John Piper:
The Undaunted Message of Freedom [Desiring God Blog] 2013-10-28 11:47

“I think, therefore I am.” René Descartes penned this popular line in his 1637 treatise, Discourse on the Method, and it’s a good summary of the book itself. Heralding the importance of scientific discoveries and the necessity of doubting one’s own view of the world, Descartes’ work is often considered the first domino to fall in what is now called the Enlightenment. The Enlightenment, with its explosion of scientific discoveries and philosophical arguments, had one task: to replace authority and tradition with reason and self-sufficiency.
For centuries, the existence of a divine being beyond our realm was largely assumed. When people talked of events in their lives such as miracles, disasters, and everything in between, they spoke of it as the will of some divine being. They were always looking vertically for their answers to life’s most essential questions. The Enlightenment, however, proposed that we must look horizontally at what is observable around us for such answers. Many of the questions that were settled for so many years became fair game in the eyes of the “enlightened” culture.
In regards to Christianity in particular, the Enlightenment hastily rejected any talk of divine authority or supernatural occurrences. While Christians believed so that they could understand reality, Enlighteners demanded that they only believe what they could understand. This seismic shift in worldview permeates our culture today, leaving Christians uneasy about the world and the world uneasy about Christians.
So although Enlightenment brought distrust for the authoritative claims of Christianity, we as Christians believe that we are servants of a God who has defined what is objectively true. What God says, who he is, is true reality for all people forever, whether a person accepts it or not.
But for the modern mind, this is foolish. Truth is not objective, but subjective, they say. In other words, it is constantly changing as we gather more data and learn more about the universe around us. Truth is often defined as what is best for the individual. If one man thinks it is acceptable to marry another man and you don’t, that’s your opinion. Your truth versus mine. And you can’t tell me otherwise. That’s the great escape of modern thinking.
To society, it is laughable that Christians gladly call themselves “servants” of God. Submitting to such an overarching authority is imprisonment, they must think. Christians are shackled by the very One who claims to set them free. Beyond all this, there is simply no observable, factual evidence that this God even exists. You can see their problem, right?
But for the Christian, being a follower of Christ is much more than adhering to a factual statement. We don’t simply say, “I’ve weighed all of the evidence and believe that the highest probability is that the Christian God exists.” That may be part of it, no doubt. But the hearts of believers experience more than a mental assent — there is an inner burning for the glory of the God who saved us. The apostle Paul tells us this in 2 Corinthians 4:3–6.
We are not simply fools chasing a unicorn through fields of four-leaf clovers. We have received “the knowledge of the glory of God.” This is why God’s authority is not an affront or an attack on our freedom. God has revealed himself to us through Jesus, and through Jesus we see that true freedom exists in submission to him in whom are pleasures forevermore (Psalm 16:11).
The Enlightenment wasn’t entirely bad. In many ways, its focus on the mind should both embolden and humble us. In an age where God is not a foregone conclusion, we are driven to take Peter’s words seriously when he warned us to be ready to respectfully and gently give a defense for our hope (1 Peter 3:15). And we are reminded that we cannot simply sit on the sidelines and hope that people show up to our worship gatherings. We must humbly go, both to the ends of the earth and across the street.
The effects we feel today from past ideological shifts are neither a surprise nor a roadblock to God. He is in control. As such, the gospel frees us to abandon both fear and self-righteousness, to push aside gimmicks and coercion. No brow-beating or salesmanship required. We carry with us the message of reconciliation (2 Corinthians 5:18–19). We get to join the mission of a God “who desires all people to be saved and to come to the knowledge of the truth” (1 Timothy 2:4).
As the truly enlightened citizens of the Kingdom, we get to marvel as God shines his light into the darkness of lost hearts, just like he did our own.
Related resources from John Piper:
What Is the Christian Gospel? (article)
God Strengthens Us by the Gospel (sermon)
The Gospel in 6 Minutes (video)
When Jesus Haunts Your Halloween [Desiring God Blog] 2013-10-28 01:00

Unclean spirits stir. Demonic thrones and dominions gather. Cosmic powers over this present darkness come to attention. And the devil himself, ready to devour and destroy, ignites his fiery darts and stretches his legs for the lion’s prowl.
As All Hallows’ Eve draws nigh, the spiritual forces of evil align, and Satan prepares his hordes for the party of the year — the grand harvest festival, celebration of darkness and death, when they pretend to be their strongest.
Halloween is almost here. But so is their final defeat. Jesus haunts their Halloween.
As the demonic rulers and authorities make ready, the one who sits in the heavens laughs (Psalm 2:4). To him, the devil is no threat, with all his orcs and goblins and the wickedest of witches. This is no evenly matched bout. If the incarnate Christ, in his humblest state, commands unclean spirits and they obey him (Mark 1:27) — how much more the risen and glorified Lord? Jesus does the real haunting.
Even as his adversaries marshal their best, they can’t escape serving his purposes. It is all through him and for him. “By him all things were created, in heaven and on earth, whether thrones or dominions or rulers or authorities — all things were created through him and for him” (Colossians 1:16). Jesus haunts their Halloween.
No demon lurks apart from his will. No spirit pounces apart from his plan. He is sovereign over even the movements of evil minds. “God has put it into their hearts to carry out his purpose by being of one mind and handing over their royal power to the beast, until the words of God are fulfilled” (Revelation 17:17).
Luther nailed it — one little word shall fell them. Jesus haunts their Halloween.
It is precisely when the devil feigns to be his fiercest that Jesus delivers the deathblow. It was a Halloween-like gathering of ghouls and goblins at Golgotha when “he disarmed the rulers and authorities and put them to open shame, by triumphing over them” (Colossians 2:15).
Jesus came to conquer fear, to haunt whatever haunts. “The reason the Son of God appeared was to destroy the works of the devil” (1 John 3:8). He stooped to share in our flesh and blood “that through death he might destroy the one who has the power of death, that is, the devil, and deliver all those who through fear of death were subject to lifelong slavery” (Hebrews 2:14–15).
Those who are in Christ have no need to fear the night. This is now our day. He has won it for us, and will not leave us to fend for ourselves in the devil’s domain. God “has delivered us from the domain of darkness and transferred us to the kingdom of his beloved Son” (Colossians 1:13). This we know: “he who is in you is greater than he who is in the world” (1 John 4:4).
“Take heart; I have overcome the world,” he says (John 16:33). Every inch of this universe — every single one — is his. And that includes All Hallows’ Eve and all its worst. He is the one who empowers us to “withstand in the evil day, and having done all, to stand firm” (Ephesians 6:13). And he says that just as he squashed the Serpent’s skull with his heel, so “the God of peace will soon crush Satan under your feet” (Romans 16:20). Our feet. Get your boots.
Jesus haunts their Halloween. And so too he must haunt ours.
When Jesus haunts our Halloween, we “put on the Lord Jesus Christ” (Romans 13:14) and “put on the new self” (Colossians 3:10). Dressed in the full armor of God, we “stand against the schemes of the devil” (Ephesians 6:11) on the exact night when he’d most want us to circle the wagons. We have a Book and will “not be outwitted by Satan; for we are not ignorant of his designs” (2 Corinthians 2:11). We take up the shield of faith “with which you can extinguish all the flaming darts of the evil one” (Ephesians 6:16).
When Jesus haunts our Halloween, we pour in the extra energy and creativity to capitalize on this opportunity to meet new neighbors and go deep with the old — whether we’re ushering our kids from house to house or leaving our lights on and giving out the best candy.
When Jesus haunts our Halloween, we remember that our enemy is not the scariest-clad Halloween reveler, but “the god of this world” who has blinded their minds and keeps them “from seeing the light of the gospel of the glory of Christ” (2 Corinthians 4:4). We war not against unbelievers but “the prince of the power of the air, the spirit that is now at work in the sons of disobedience” (Ephesians 2:2). We wrestle not against flesh and blood, but the rulers, authorities, cosmic powers over this present darkness, and spiritual forces of evil in the heavenly places (Ephesians 6:12).
When Jesus haunts our Halloween, we look on the cheekiest carousers with compassion — as “harassed and helpless, like sheep without a shepherd” (Matthew 9:36). On this night, as much as any, “the harvest is plentiful, but the laborers are few,” and so we “pray earnestly to the Lord of the harvest to send out laborers into his harvest” (Matthew 9:37–38). And we walk in faith to be those workers.
And when Jesus haunts our Halloween, we fight not only Satan, but fear in our souls. We see that our Halloween horrors reveal our lack of faith in who Jesus is, what he has accomplished, and that he has commissioned us.
When Jesus haunts our Halloween, we do not flee, but go on the offensive. “Resist the devil, and he will flee from you” (James 4:7). We don’t retreat, but resist — with level heads and open eyes. “Be soberminded; be watchful. Your adversary the devil prowls around like a roaring lion, seeking someone to devour. Resist him, firm in your faith” (1 Peter 5:8–9). We engage, with care and with courage.
When Jesus haunts our Halloween, we remember that the forces of evil, which we can be so prone to fear, are actually terrified of Jesus. Everyday is a spook for the devil and his demons, and Jesus does the haunting. The decisive blow has been dealt, and soon we will land the final punch.
Jesus has promised his gospel will advance (Matthew 24:14). He will build his church, and the gates of hell will not prevail (Matthew 16:18). And so when Jesus haunts our Halloween, we join the triumphant anthem:
“O death, where is your victory? O death, where is your sting?” The sting of death is sin, and the power of sin is the law. But thanks be to God, who gives us the victory through our Lord Jesus Christ. (1 Corinthians 15:55–57)
More on Halloween from Desiring God:
Three Prayers for Facing Monday (Or Any Tomorrow) [Desiring God Blog] 2013-10-27 01:00

This is one of those really deep, common truths — one which Jonathan Edwards expounds with the intellectual horsepower of a genius, and to which our most common experience testifies:
Essential to our present joy is the anticipation of greater joy to come.
This is why, for example, the best part of going on vacation is often the day before we start it. The glad anticipation of what will be compounds in the present and gives us a good feeling. But the closer we get to the last day of vacation, the more the joy diminishes. Sound familiar?
In American culture, the weekend can be a miniature version of this experience. After five days of work, many of us look forward to two days off on Saturday and Sunday. The height of anticipation comes Friday — TGIF! — but by Sunday evening the cheer is gone. Tomorrow we face Monday, with all its certain trials and trying uncertainties.
So how will you face it? How can we make the most of Sunday to prepare for the less-than-enthusiastic tomorrow?
In complement to corporate worship, here are three prayers for facing Monday:
1. Give me a brazen trust in your greatness and your goodness.
Whatever circumstances may come our way tomorrow, the most foundational truth we need to know is that God is in control, and that he is good. Many of us can recite the dinnertime prayer “God is great, God is good…” — but we need more than a good memory for this fact to take effect. We need faith. We need a brazen trust — an indomitable confidence — that our God rules the kingdom of men, that no purpose of his can be hindered, that all he pleases to do he does (Daniel 4:17; Job 42:2; Psalm 115:3). And that he abounds in steadfast love, that he is compassionate and merciful, that his nearness is our good (Exodus 34:6; James 5:11; Psalm 73:28).
Saying it is one thing; believing it is another. So we ask God for this faith.
2. Give me a humble heart towards the people I will encounter.
Most circumstances we face involve faces. Real people. People with their own stories. People with eternal souls. This means oftentimes how we face situations is really about how we relate to others. And what we need is humility. We need a deep, sincere sense that we are creatures. If the first prayer is to know the greatness and goodness of God, this second prayer is to know that greatness and goodness are original to him, not us. We are not that great. We are not that good.
Admitting this doesn’t come natural. So we ask God for this heart.
3. Give me the deep joy that because of Jesus, the best is always yet to come.
This is no cliché. No too-good-to-be-true platitude. For the Christian, the best is always, always, yet to come. The first two prayers come together in this one: a great God will judge all evil, a good God will show mercy, and Jesus vividly showed both for the helpless.
On the cross, Jesus simultaneously absorbed God’s wrath for sinners and demonstrated God’s love for sinners (Romans 3:25; 5:8). And because he did this, because we are united to him by faith, no circumstance in this life is worth comparing to the glory that will be revealed to us. The best is always yet to come. Even in eternity, as Edwards explains, we will never stop saying this.
And that is reason for unwavering celebration. So we ask for this deep joy.
Recent posts from Jonathan Parnell:
Preachers, Find Your Voice [Desiring God Blog] 2013-10-26 01:00

Christian preaching is not parroting.
As desirable as it is to copy a skilled communicator, and as unavoidable as it is to imitate those who have shaped us most, there is good reason for a preacher to find his own voice. Not vanity, but being true to what Christian preaching is.
Before it is heralding a message, preaching means first and foremost stewarding a message. Before we “preach the word” (2 Timothy 4:2), we should be devoted and unashamed students, “rightly handling the word of truth” (2 Timothy 2:15). Before telling others what God has to say, we must hear his voice ourselves and deeply know his speaking.
This relationship between studying and preaching, between stewarding and heralding, gets at the heart of what it means to be a pastor in the church age, the time between Jesus’s ascension and return.
Throughout history, God has used spokesmen. Long ago, at many times, and in many ways, he spoke through the prophets, but in these last days, he has spoken to us in his Son (Hebrews 1:1–2). The apostles were the Son’s inspired spokesmen in their day, and still are in their writings. And now in the days of the church, a major way in which God’s word is re-revealed to his people is through the regular, ordinary, week-in, week-out ministry of local church pastor-preachers. This is a distinct season in redemptive history in which God’s heralds have been entrusted with “the whole counsel of God” (Acts 20:27) and with specific flocks to lead and feed.
This preaching particular to the church age is what Jason Meyer calls “pastoral preaching.” In Preaching: A Biblical Theology, he provides a whole-Bible picture of how God has worked through his spokesmen across history to bring his word to his people — and especially how that relates to preaching in the local church today.
In this new episode of Theology Refresh, Meyer challenges us to consider the task of preaching as first stewarding God’s revelation, and then heralding that revelation in such a way that the church encounters God in his word. Preachers are called to say to their flocks what God has to say, show their flocks where it comes from in the Bible, and shepherd their flocks where it leads. And, yes, it most emphatically does mean preaching the Old Testament in a distinctly Christian way — and relentlessly bringing gospel-centrality to bear in the pulpit.
To access this 12-minute episode, subscribe to Theology Refresh in iTunes, listen or watch at the resource page, download the audio, or watch below.
For more on Preaching: A Biblical Theology, see Desiring God’s 1-minute video with Meyer.
Recent episodes of Theology Refresh:
To subscribe or see the full list of almost 50 episodes, visit Theology Refresh in the iTunes store.
What to Neglect to Have a Rich Life [Desiring God Blog] 2013-10-25 01:00

Let the word of Christ dwell in you richly, teaching and admonishing one another in all wisdom, singing psalms and hymns and spiritual songs, with thankfulness in your hearts to God. (Colossians 3:16)
This verse from Colossians is so full of nourishment that there is no way to put the whole thing in our mouths at one time. It’s going to take a few blog bites to chew on it.
Today, all I want to do is chew on the first word: “let.” Let the word of Christ dwell in you richly.
Another way to say it is, don’t stop the word of Christ from filling you to satisfaction. Or stop stopping it.
Here’s the thing: we are frequently impoverished spiritually by our own not letting ourselves be rich. On our shelves or bed stands or in our tablets or computers is a bank vault of “true riches” (Luke 16:11). But the pawnshop trinkets of worldly words are deceptively attractive. We can even be on our way to spend our time (the currency of life) on the riches in the vault and end up spending it in the pawnshops along the way.
What Paul wants us to do is neglect things that make us poor and not neglect things that make us truly rich.
If the word of the Wall Street Journal or World Magazine or Wired Magazine or David Brooks or David Letterman or David McCullough, or John Mayer or John Steinbeck or John Paul II or John Calvin or Richard Dawkins or Richard Branson or Richard Baxter or Bono or Bach or blogs (even this one) dwells in you more richly than the word of Christ, you’re poor. You might be impressive at a dinner party or around a conference table or at small group. But you’re poor. You’re storing up dust.
You don’t need to be in the know.
You don’t need to be admired among the literati or respected in the guild. You don’t need an impressive net worth. You don’t need to be well traveled or well read. You don’t need to be conversant in Portlandia or know how many Twitter followers Taylor Swift has. You don’t need to be politically articulate, or up on the mommy blogs or the young, restless and reformed buzz. You don’t need to see the movie. You don’t need to read the novel. You don’t need to look hip.
But what you desperately need, more than anything else in the world, is the word of Christ dwelling in you richly.
No one speaks like Jesus Christ (John 7:46). He is the Word of God and the Word that is God (John 1:1) He is the Word of Life (1 John 1:1) and when he speaks, his word is living and active (Hebrews 4:12) and he shows you the path of life (Psalm 16:11) and his words give you hope and joy and peace (Romans 15:13).
Jesus is the one human being in all of history who speaks the very words of eternal life (John 6:68) and when you listen and believe his word, it becomes your life (Deuteronomy 32:47), your food (John 6:51), your drink (John 4:14) and your light (Psalm 119:105).
Only Jesus has the words of life. Only him. That’s why the Father pleads with us, “This is my beloved Son; listen to him” (Mark 9:7).
Everyone else’s words are dust in the winds of time and to chase them is to chase the wind (Ecclesiastes 1:14). The precious few helpful, enlightening, even mortal life-preserving words are only of superficial help to us and in the end will blow away.
The only exceptions are those that help us (and others) listen to the word of Christ.
Let the word of Christ dwell in you richly. Don’t neglect it. Listen to his word. Soak in his word. Memorize his word. Eat and chew it slowly. Don’t stop it from benefitting you.
Neglect the TV, blogs, social networks, video games, theaters, magazines, books, hobbies, chores, and pursuits that keep you from the Vault. Neglect the impoverishing pawnshop trinkets of words that will turn to dust in a day, a week, or a few years.
When it comes to life, time really is money. Time is how you spend your life. Don’t waste it. Spend your best time buying “true riches.”
Recent posts from Jon Bloom:
15 Quotes from ‘Jesus > Religion’ [Desiring God Blog] 2013-10-24 11:30

Jefferson Bethke has recently published Jesus > Religion: Why He Is So Much Better Than Trying Harder, Doing More, and Being Good Enough. The book’s title and content are inspired by his spoken-word video, “Why I Hate Religion, But Love Jesus,” which has been viewed 26 million times. Yes, 26 million.
Filled with Jefferson’s own story, the book is raw and authentic. It will engage many, especially young people, who have been hurt by or given up on the church. His conviction and passion are contagious and inspire hope for the next generation of Jesus-lovers and leaders. In the end, Jesus > Religion is about grace — unconditional, scandalous, glorious grace. And we all — every one of us — could use more of that.
We wanted to give you a flavor for the book, so we pulled our fifteen favorite quotes.
“I heard enough sermons to know Jesus died for me, but I also had such a broken and painful life that I figured Jesus wasn’t relevant… I had just enough Jesus not to need him at all.” (3)
“Initially, I blamed God for the pain in my life, but slowly I started to hear the whisper of his grace. I didn’t know it then, but God broke me to fix me because he loved me.” (6)
“Grace isn’t there for some future me, but for the real me. The me who struggled. The me who was messy. The me who was addicted to porn. The me who didn’t have all the answers. The me who was insecure. He loved me in my mess; he was not waiting until I cleaned myself up.” (7)
“Be careful when you pursue truth, because you just might find him.” (35)
“Heaven isn’t a place for people who are scared of hell; it’s for people who love Jesus. The reason heaven is heavenly—full of joy, life, and bliss—is because we’ll be with Jesus.” (44)
“If you care more about flaunting your Christian freedom than promoting Christian unity, you’re probably not free. You are actually a slave to your so-called freedom.” (53)
“No one is more religious than the Christian who gives grace to everyone except the religious older-brother types. [God] gives grace to the younger and the older. No one is past redemption. No one is past grace. All God wants is for both the religious and the rebellious to come into the party.” (56)
“The truth is, God doesn’t grade on a curve; he grades on a cross… A grace economy is backward to most of us—those who think they qualify, don’t; and those who admit they don’t qualify, do.” (78-79)
“[The Bible] was not given to us so that we could highlight and underline our way into eternity, but in hopes that we would have a special encounter with our Creator.” (85)
“God’s ways aren’t our ways, but his ways save. Our ways don’t.” (89)
“When we become Christians, we begin to follow Jesus, but the moments when he completely obliterates our self-righteousness and gives us a potent dose of real, transforming grace is when following him becomes deeply special.” (132)
“That’s the truth with God’s grace. It’s not that we are holding on tight in hopes to not be seduced by our old life and sin, but rather it’s that God’s grace is so sweet and precious it compels us to stay with it. Grace is better music than sin.” (152)
“Our lives on earth aren’t just placeholders until we go to heaven. We are to create, cultivate, and redeem while we’re here… We are created to infect and infiltrate culture, restoring and reclaiming what is God’s.” (156, 172)
“The truth is, God doesn’t just want your ‘Christian’ things. He wants it all. When we realize the beauty of God’s grace in the mundane, not just the religious, that’s when we will begin to see him correctly.” (166)
“I saw that the church wasn’t a museum for good people; it was a hospital for the broken. Jesus wasn’t trying to create a place to show off his shiny employees; he wanted a place where his children could be healed.” (186)
Quotes are from Jefferson Bethke’s Jesus > Religion. Related resources from Desiring God:
Single, Satisfied, and Sent: Mission for the Not-Yet Married
20 Quotes from The Explicit Gospel (Chandler)
Don't Waste Your Life (Piper)
Five Facts About Loving God [Desiring God Blog] 2013-10-24 01:00

What is the relationship between loving God and neighbor, and how can both Jesus and Paul say that loving our neighbor fulfills the law (Matthew 7:12; Romans 13:8; Galatians 5:14)? Isn’t love for God an even higher priority?
Moses helps us answer these questions in Deuteronomy 10:16–19, where he portrays a radical love of neighbor as the key test to measure whether we are loving God with all.
With an echo of the call to love God with all, Moses opens Deuteronomy 10 by calling Israel to maintain radical God-centeredness (Deuteronomy 10:12–13). Yahweh is always to be the blazing center in his people’s solar system. He then notes that such wholehearted, life-encompassing allegiance to God was warranted from Israel because he created them and because he rescued them from Egyptian slavery (Deuteronomy 10:14–15). In light of these truths, Moses then applies the call to radical love for God into Israel’s everyday lives, and in the process, he reveals how far they were from God’s ideal. I see five significant points regarding love for God in these verses.
Moses first charges Israel to “circumcise the foreskin of your heart, and be no longer stubborn” (Deuteronomy 10:16). Israel was hardhearted, and hardhearted people cannot love God. Indeed, their hardness went deep, controlling the core of their very identities. As Moses said in the previous chapter, “You are a stubborn people. . . . From the day you came out of the land of Egypt until you came to this place, you have been rebellious against the LORD” (Deuteronomy 9:6–7). Until their hearts got fixed, love would not be evident.
The reason why hardheartedness is such a problem is because God rightfully demands all our allegiance, and any hardness toward him is an idolatry problem. “For the LORD your God is God of gods and Lord of lords, the great, the mighty, and the awesome God” (Deuteronomy 10:17). Yahweh is the only God in the pantheon of heaven (Deuteronomy 5:7; 6:4), and therefore he alone holds the right to our absolute surrender (Deuteronomy 5:8–10; 6:5). He alone is the preeminent savior, sovereign, and satisfier, and therefore misplaced affections are foolish and suicidal. God is to be the sun in our solar system, not one of the planets circling us. Idolatry separates us from love.
It is at this point in Moses’s sermon that we begin to see more clearly how intimately he tied the call to love our neighbor with the call to love God. Indeed, implied in the text is that those who have hearts of love toward God will ultimately begin to resemble God himself, who “is not partial and takes no bribe. He executes justice for the fatherless and the widow, and loves the sojourner, giving him food and clothing” (Deuteronomy 10:17–18).
Idolatry of the heart is seen in a failure to love as God loves. Love for God is displayed in whether we are willing to love our neighbor, even those who are most difficult to love –– to sacrifice our time, treasures, and talents for the sake of those whom God loves.
In my own life, I feel that only recently have I begun to understand what it means to love God in this radical neighbor-love way. Over the last five years, my family has journeyed the road of trans-racial, international adoption, seeing the curse of orphan status broken in the lives of three little treasures. By faith the saints of old “were made strong out of weakness, became mighty in war, . . . were tortured . . . suffered mocking . . . of whom the world was not worthy” (Hebrews 11:34–38).
Radical neighbor-love magnifies the worth of God, regardless of the cost, and displays the type of love God himself has shown. “Whoever would be great among you must be your servant . . . even as the Son of Man came not to be served but to serve” (Matthew 20:26, 28). “Those who are well have no need of a physician, but those who are sick. Go and learn what this means, ‘I desire mercy, and not sacrifice.’ For I came not to call the righteous, but sinners” (Matthew 9:12–13). May God help us love this way.
Having identified the hardness, idolatry, and godlessness of his hearers, Moses now urges them to love the broken –– to see their hearts surrendered wholly to Yahweh’s supremacy, manifest in care for the needy: “Love the sojourner, therefore, for you were sojourners in the land of Egypt” (Deuteronomy 10:19). Loving God with all is about loving others as we ourselves have been loved. Israel knew what neglect, abuse, emptiness, hunger, and poverty were like; they had experienced all these in their past. They knew what it meant to be dirty, and they knew what it meant to be redeemed. Believers today have experienced an even greater redemption through the victorious work of Christ Jesus. Sins are forgiven, and righteousness is won. And “if God so loved us, we also ought to love one another” (1 John 4:11).
Radical neighbor-love is birthed in the soul of one who has tasted the sin-overcoming, mercy-saturating, joy-filling love of God, now manifest in the person of Christ. “We love because he first loved us” (4:19). Loving the broken is overflow from the life that has been filled with love from God. “If anyone says, ‘I love God,’ and hates his brother, he is a liar; for he who does not love his brother whom he has seen cannot love God whom he has not seen” (1 John 4:20).
Many recognize how difficult it is to pour out our own resources in love for the hurting, the weak, and the marginalized. Love for the broken is messy and wearying and unattractive, apart from the amazing grace of God. In the end, this kind of radical neighbor-love is impossible without God’s help (Deuteronomy 29:4), and therefore it becomes a key test for knowing whether we are connected to the source of love.
More from Jason DeRouchie:
A Beer with Jesus? [Desiring God Blog] 2013-10-23 11:13
Is it a sin for me to drink alcohol?
It could be, says Pastor John in today’s episode of the Ask Pastor John podcast.
“I’m a default teetotaler,” he says. “And what that means is if I have my choice, I don’t drink alcohol, but I might, to be a good guest. . . . But I don’t think anybody can make a case from Scripture that teetotalism is required.”
So what’s his case? Pastor John explains in today’s podcast (episode #200):
Also, this week we introduced Ask Pastor John podcast videos to YouTube — yes, videos. But if you’re expecting video footage of Pastor John answering questions, you’ll be sorely disappointed. We have taken the audio recording, set it to a photo, and made it into a video to reach new audiences of YouTubers. Find those available videos here.
As an example, here’s how episode 79 — “How to Fight Laziness” — appears:
Ask Pastor John is a daily podcast series of 3–8 minute conversations released each weekday at 10:30am (EST) through the DG Facebook and Twitter feeds. You can tune in to the new episodes through the free Ask Pastor John mobile app for iPhone and Android. We’re currently hosting all the recordings on SoundCloud, a website making it easy to listen to several of the podcasts in one sitting. They’re also archived on the DG website, syndicated in iTunes, and select episodes are being adapted to video and offered in DG’s YouTube channel.
We want to hear from you. To submit a question to Pastor John please include your first name, hometown, and brief question via email: AskPastorJohn AT desiringGod DOT org.
Thanks for listening to the podcasts. We appreciate your engagement and interest.
Simple Greatness: The Story of My Hmong Father [Desiring God Blog] 2013-10-23 07:17

How is the gospel real to you? This was a question that I was asked in college.
As part of a campus ministry, I heard the word “gospel” thrown around a lot, but as for how it was real to me, that wasn’t as easy to explain. The short answer was that the gospel is real to me because it’s how God saved me. The truth of the gospel is alive to me because by it God made me alive. But as I looked closer at God’s work in my life, I began to notice a “tangible remnant” of Jesus’s love for me. It’s embedded deep in my story as a Hmong-American. Let me explain.
If you’re familiar with the Twin Cities of Minnesota, you’ve heard about a people group called the Hmong (the H is silent). We are a group of war refugees that immigrated to the Midwest in the late 1970s and early 80s, because of the Vietnam War. In the Twin Cities metro, there are over 66,000 Hmong people — the largest concentration of Hmong in the United States.
The Hmong are an agricultural people group, known as the “hill tribe,” originating from the mountains of Laos. Some historians even trace them back to western China. Many of the Hmong people were persecuted by the surrounding Laotians and Thais because of their independent way of life and unwillingness to conform to the dominant society. In fact, the reason the Hmong people lived among the mountains is because they wanted to be independent, even though it’s a harder agricultural lifestyle when compared to the fertile low land.
During the Vietnam War, the U.S. government allied with the Hmong because of the Hmong’s strong anti-communist views. Hmong men were trained to conduct hit-and-run tactics against the North Vietnamese Army (NVA), guard secret U.S. radar stations and bases, and rescue American pilots shot down over Laos and Vietnam. A promise was made that anyone who fought with the Americans would get a free pass to the States if the war was lost and the Hmong no longer had a home. Many of the young Hmong men joined up and started fighting against the communists, that is, the NVA and local Laotian communist groups.
My father was one of these men.
My father and his brothers were recruited by the CIA and trained in guerrilla warfare. They were paid a small wage and given weapons to fight for the United States.
After the Fall of Saigon in 1975, the American troops moved out of Vietnam and all of South East Asia, including the villages of their Hmong allies in northern Laos. Left completely vulnerable to the communists, many Hmong people were hunted down and killed. The communist soldiers invaded one village after another, killing the children, raping the women, and torturing the men to death. Many were sent to “re-education camps” and were never heard from again.
Some Hmong people were given asylum in the States, but the majority of them where left behind to fend for themselves. To escape this genocide, the Hmong people started fleeing west towards Thailand. Parents carried their children into hiding and slowly made their way through dense jungle, until they came to the Mekong River (the Mississippi River of Southeast Asia). Crossing the Mekong River had to be done at night, in the dark, with makeshift floatation devices from bamboo. Many Hmong people drowned on this river because of enemy attacks, the strong current, and harsh conditions. (Both my parents lost their spouses at the Mekong crossing and were widowed when they met each other in Thailand.) If they crossed the river, then they would be able to settle in the refugee camps in Thailand. That is where my siblings and I were born, and where I spent the first five years of my life.
Since my father fought for the United States, it was easier for him to get a pass to the country. I still remember going through customs and getting my picture taken for my residential alien card. I was entering a brand-new world. It wasn’t until about seven years ago that my grandmother told me the whole history of my father back in Laos.
He never talked about it, but my father, in the midst of intense hardship, was a leader in his village. He had a commanding presence and was highly respected by the other men. He would have been considered as something similar to a mayor.
But when the opportunity came for him to move his family to America, he left it all. Away from his work, away from his comfort, away from his high social status — my father walked away from it all to live in a country whose language he didn’t speak and whose culture he didn’t know. He abandoned his authority, and the respect he had won, to welcome jobs and endure insults sadly typical for a middle-aged immigrant in America who doesn’t speak English.
And he did it so that his children would have a better life. I didn’t know what I needed. I didn’t know the extent of the danger around me. But my father became nothing so that my siblings and I could live in peace.
My father has worked hard to provide for us, but even more than that, he has fought for our family as our spiritual leader. When I was in high school, I remember waking up early one morning to find him kneeling in his dirty work clothes, praying for each of his children by name. That image has stayed with me since, and I would learn that he and my mother repeated this routine everyday before he left for a long day of work.
The message of the gospel is that we need a Savior. We’ve turned away from God in our sin and deserve his wrath. But Jesus, though he was rich, became poor for us so that we, in his poverty, might become rich (2 Corinthians 8:9). He left his status so that we might know a new one: righteous, forgiven, at peace with God.
So how is this real to me? How do I stay alive to the gospel’s wonder?
Everyday I see glimmers of the gospel’s light in my father. He is a simple man in American terms. He still doesn’t speak English. He doesn’t have an education. He works hard with his hands to earn a living. But for three decades, he has preached the gospel to me with his words, and exemplified it with his life. He laid his life down for my siblings and me, which as Jesus says, is the way that he loves, and the greatest kind of all (John 15:12–13).
Related resources from John Piper:
What Is the Christian Gospel? (article)
God Strengthens Us by the Gospel (sermon)
The Gospel in 6 Minutes (video)
A Whole World Hangs on a Word [Desiring God Blog] 2013-10-22 01:00

Sometimes a whole world — a whole theology — hangs on a word.
Consider the word “this” in Ephesians 2:8. Does it refer to “faith” or “grace” or both? Is faith a gift of God?
For by grace you have been saved through faith. And this is not from you; it is the gift of God.
What does “this” refer to? “And this is not from you; it is the gift of God.” What is its antecedent? The question is not settled by the fact that in Greek “this” is singular and neuter, while “grace” and “faith” are both feminine. “This” is just as ambiguous in Greek as it is in English.
But consider these four pointers to seeing faith as a gift in Ephesians 2:8.
1. When Paul says “this is not from you, it is the gift of God,” he seems to be referring to the whole process of grace-faith-salvation. That may be why “this” is neuter and not feminine.
2. But more important than that is the way Paul uses the phrase “by grace you have been saved” back in verse 5. In verse 8, he says, “For by grace you have been saved through faith.” Back in verse 5, he said, “When we were dead in our trespasses, [God] made us alive together with Christ — by grace you have been saved — and raised us up with him.”
This is striking. Paul breaks the flow of his sentence in order to insert “by grace you have been saved.” And he does it precisely after saying, “When we were dead, God made us alive.” Why does he insert “by grace you are saved” just here?
Is it not because he wants to make clear the true nature of grace? He made you alive when you were dead — by grace you are saved! This grace is God’s free act of giving life to the dead. By inserting “by grace are you saved” immediately after saying “when you were dead, God raised you,” he shows that this saving grace is not caused by our participation. We are dead when it happens to us. This saving grace is resurrection of the dead.
So when Paul gets to verse 8, one of the reasons he repeats, “By grace you have been saved,” is to describe how we experience this divine miracle of being raised from the dead. He adds “through faith.” “For by grace you have been saved through faith.” In other words, the life God creates by grace out of death is experienced in our believing. Our believing is what this new life does that grace creates. So faith is the creation of grace. Therefore, it is part of the gift in verse 8 that is not from ourselves.
3. This is confirmed in verse 10 when Paul actually uses the language of “creation” to describe our new life as believers: “For we are his workmanship, created in Christ Jesus.” The language of “creation” confirms that when God “made us alive” (verse 5), we were not part of the cause.
Things that are created do not cause their creation. The existence of a new believer is a “creation in Christ Jesus.” And that confirms that this new believing is part of the gift in verse 8. Our faith is a gift of God.
4. Finally, consider that Paul says the same in Philippians 1:29 — that our faith is a gift: “It has been given to you that for the sake of Christ you should not only believe in him but also suffer for his sake.” Literally: “It has been given to you to trust him.”
So when Paul says, “By grace you have been saved through faith, and this is not from you, it is the gift of God,” part of his meaning is that our faith is a gift of God. It is a divine creation. It is the work of grace when we were dead. It is not “from ourselves.” Therefore, our faith is the mark of being chosen by God. He chose to give us faith.
A whole world — a whole theology — hangs on a word. “This is not from yourselves.” “It is the gift of God.” That is, faith is not from yourselves. Faith is a gift of God.
To believe this changes everything. You live in a different world if you believe this. We will be discovering the wonders of this world for all eternity.
Recent posts from John Piper:
Our Children for Our Joy [Desiring God Blog] 2013-10-21 11:31

I dropped my son off at his school and yelled my usual through the rolled down window, “I love you. Make good choices. Obey your teacher.” As I began to roll up the window and drive away, my little first grader took his small hand to his mouth and blew me a kiss.
It was like everything stopped at that moment.
I realized how quickly this season would last. Would he blow me a kiss when he’s 16 years old? I don’t know. I blew him a kiss back and he waved to me, mouthing the words “Bye, Mom.” I was overwhelmed. I wished I could freeze that point in time.
I like to call my children sweet ragamuffins. Motherhood is challenging. My kids don’t obey me every time I ask them to do something. They are rambunctious, loud, and messy. And they are sweet. They are gifts. Like many moms, I wouldn’t trade it for anything. What I think we can so often forget, though, is that motherhood isn’t a task to be checked off like laundry. It is a calling.
Maybe the word “calling” makes you want to run and hide. For many, “calling” can sound as if motherhood is your only identity, that is all encompassing and you never get a break from your endless responsibilities. This is not true. You are likely called to be a wife and church member and friend as well (and the list could go on). So motherhood is not your only identity; it is a part of your identity. And there is a weight to that. Mothers are more than just mothers, but we are never less. God’s word instructs us to train up our children in the way they should go (Proverbs 22:6). I can’t think of a greater challenge given to us parents. As one in the throes of raising and teaching young children, I am desperate for Jesus.
But I don’t think remembering the responsibility to train our children is the best way we embrace and savor these short days we have with them. Remember that “every good and every perfect gift is from above, coming down from the Father of lights…” (James 1:17). Our children are not tasks to complete, but gifts to enjoy. And we do this by remembering that they are truly gifts from God. Yes, even when they stand in the hall refusing to put away their socks, or when they throw their cereal on the floor, or when they make it almost impossible to complete a trip to the grocery store. Those are trials mothers face weekly and yes, even they are gifts.
Paul, instructing Timothy to challenge the rich to put their hope in God instead of their wealth, reminds us that it is God who provides all things for our enjoyment (1 Timothy 6:17). Our children aren’t meant to be checked off a list. They are to be delighted in. As with every gift we receive, we must be careful not to idolize our children. Only God should be worshipped. But what if we began to think of our kids as true gifts from God aimed at our enjoyment, both in our kids and in God through them.
I think of how much I enjoy looking at colorful birds at the zoo. They are exotic creatures, each with their unique beaks and beautiful mosaic of feathers. They are a wonder of God’s creation, and he cares for them. But not more than he cares for us: “Look at the birds of the air: they neither sow nor gather into barns, and yet your heavenly Father feeds them. Are you not of more value than they?” (Matthew 6:26).
In a similar way, I can think of many things I enjoy, but I value my kids more. I love looking into my kids’ precious eyes. I want to get into the world of their God-given personalities and take in their laughs and answer their questions. I want to enjoy them.
Maybe that’s precisely what the main thing of this mommy calling is all about. Maybe it’s not as much a call to train your kids as it is a call to treasure them.
Our children won’t be our little children forever. Let’s enjoy these days that God has given us. They are his gifts, glimmers of his goodness, which leads us to say with Lewis, “What must be the quality of that Being whose far-off and momentary sparkles are like this!”
More from Grace at Home:
What the Church Can Learn from Chick-fil-A [Desiring God Blog] 2013-10-21 01:00

The cows started writing on billboards in the Twin Cities metro earlier this year. End of Burgerz — Koming Soon.
These bovines can’t spell, but we got the message — especially those of us Minnesotans who are transplants from the South. Here comes Chick-fil-A, at long last. Time to Eat Mor Chikin.
It’s no secret the chain was founded by an unapologetic Christian from the Atlanta-area. Now in his nineties, Truett Cathy has operated his restaurants on overtly Christian principles since the 1940s. His son, Dan, the franchise president, is known for his support of Christian causes and his opposition to so-called same-sex marriage, which drew national attention last year.
For the Cathy family, it goes deeper than closing on Sundays, playing Christian music, and putting “glorify God” in the corporate purpose statement. For decades, they have tried to apply the biblical worldview and ethic not just to the surface, but to press it into the culture of the chain, not only in the dining room but behind closed doors.
As I made my first visit to a stand-alone Chick-fil-A here in the Twin Cities last week, and then consulted Truett’s book, I noticed five biblical principles, among others, at work in the corporate culture — principles our local churches and leaders might benefit from being reminded of.
Let this much be clear: The church doesn’t need Chick-fil-A. We don’t need successful Christian businesses, athletes, films, and reality shows for the advance of the gospel. The tip of the spear is the local church. But when we can glean a few pointers from another body reading our Book, we might as well take notice.
This may be the key insight for Chick-fil-A over and against other franchises. From the beginning, Truett has strained to show that it’s not simply about the bottom line. “Profit is not the name of the game,” he says. “It is only the scorecard for some of our accomplishments” (How Did You Do It, Truett? 55).
The effects of this emphasis are pervasive in the company’s culture. It makes it possible to close all day Sunday, and observe the six-and-one creation principle (Genesis 2:2), when other restaurants make more than 20% of their sales this day. It encourages the leadership to focus relentlessly on long-term health and stave off the endless temptations to cut corners for short-term growth and “success.” It leads to concentration on the quality of the chain, and each individual operator and restaurant, rather than outrunning the supply lines and compromising quality for quantity of stores and sales. Which means growing more slowly — and taking so long to get to Minnesota! — rather than swelling as quickly as possible. Truett writes,
To succeed we knew we had to start small and grow slowly. This is where so many start-up companies today make their mistake. Dreamers dream big, and they want to reach their goals quickly. There’s nothing wrong with big dreams. But my experience tells me that we’re more likely to reach our dreams if we climb with care and caution, putting one foot in front of the other. To some, this may be the biggest sacrifice of all — giving up the dream of instant “success.” (17–18)
It’s a helpful reminder in the church about something we should have learned long ago. It’s not a numbers game. Discipling the nations (Matthew 28:19) is not only about quantity, but mainly quality. When well-established and constantly called to mind, this principle has a pervasive effect in the local church as well.
Truett says, “Keeping [the restroom] clean doesn’t require special skill, just discipline that comes from being concerned for the customer” (47). Put another way, Do unto others — it’s Jesus’s “Golden Rule” (Matthew 7:12; Luke 6:31), which, “You can’t beat . . . as a business philosophy.” One of Truett’s most common sayings is “Courtesy is cheap, but it pays great dividends” (35).
Sadly, some local churches drift into the mindset that the cleanliness of the facilities and the care of the grounds aren’t important — after all, the church’s real business is spiritual. But such is a failure to demonstrate Christian love and kindness. It’s a missed opportunity to consider others more significant than ourselves and look to their interests (Philippians 2:3–4) and serve.
“If you really aren’t interested in serving others,” says Truett, “you don’t need to be in the restaurant business in the first place” (37). The same is true for pastors, elders, and church staff. The church’s leaders and employees are there not to be served, but to serve.
It’s my pleasure. That’s the response you’ll often get when you thank a Chick-fil-A employee for their service. And those of us at Desiring God like to point out that it’s not just Christian, but Christian Hedonistic. It’s part of “second-mile service,” with roots in Matthew 5:41. It’s not just the Golden Rule, but more — making the extra effort and taking the initiative to serve the customer in surprising ways.
How much more should this be true in church leadership. We both aim at creating joy, and do so fueled by joy. We pastors and elders endeavor for our people’s “progress and joy in the faith” (Philippians 1:25). We are workers with our people for their joy (2 Corinthians 1:24). And we bend every effort to “do this with joy and not with groaning” (Hebrews 13:17). It’s my pleasure, as an authentic expression of a joy-filled heart of service, is a beautiful pastoral response to our people’s words of gratitude.
Fostering trust among a growing leadership team is seriously time-consuming. Without being free from a bottom-line-only focus, it’s doubtful it will happen. But such trust-building, says senior vice president Perry Ragsdale, is “the biggest key to our success” (56).
Chick-fil-A won’t take on new franchise owners just because they have the money. More important than funds is character, integrity, and trust. Which makes finding new operators a lengthy process and slows down the speed of how quickly the chain could expand otherwise. But it’s worth it in the long run. Says Truett, “The most important decision we make at Chick-fil-A is selecting restaurant Operators . . . . Our franchise Operators determine the success of the chain” (64).
But not only does the franchise carefully guard the gate, but once leaders are in, they seek to cultivate among them an openness to dialogue, discuss, and disagree. When leaders have been carefully chosen on the front end, and there’s intentional effort to grow trust among the team, asking questions and expressing concerns becomes a huge asset, rather than threat, to the organization. And so with pastors and elders in the local church.
The name Chick-fil-A has limited what the chain can do. There is plenty of room to try new things and add waffle fries, chicken nuggets, or desserts, but with chicken in the name, they won’t offer pork or beef. And that unbending commitment to what is central has produced big payoffs in the long haul with brand and mission clarity.
The Christian church is a “creature of the gospel” — created and sustained by the gospel, for the defense and advance of the gospel. The church is “a pillar and buttress of the truth” of the gospel (1 Timothy 3:15). It is the good news that Jesus saves sinners that is “of first importance” (1 Corinthians 15:3), that is our heart and center, that must be affecting and shaping all we do. The church is a veritable Gospel-fil-A.
We do more than just preach the gospel. We must. But we must do the other things in right relationship with our essential message. It is so easy to get distracted from the main thing. It’s so common for wonderful peripheral things to slowly displace the center.
Amidst all the complexities of growth and contextualizing for new locations and demographics, Chick-fil-A has kept chicken central. How much more must the church labor and strain — with great gladness and deep joy — to keep the gospel of Jesus at the very heart of who we are and all we do.
Recent posts from David Mathis:
Jesus Doesn’t Love You Like That [Desiring God Blog] 2013-10-20 01:00

Either Jesus died to save his church or he didn’t. There isn’t a third option.
Either he gave himself up for his bride, as Ephesians 5:25 tells us, or he died to create the possibility of her salvation that depends upon the skills of human decision-making.
Are we dead in our sins, as Ephesians 2:1–3 says, or are we slightly impaired? Are we “far from the peaceful shore” or are we gone, sunken to the bottom of the ocean with no chance of resuscitation? Does God toss us a floatation device, or does he raise us from the dead?
Was the cross of Christ a triumph over sin and evil, as Colossians 2:14–15 says, or was it just a nice first-move? Is Jesus victorious for the sake of his church, or did he spot us a few points? Did he suffer at Golgotha to demonstrate God’s grace to sinners, or was it a presentation of sorta-kinda-maybe hope for those smart enough to understand?
Did Jesus drain the dregs of God’s wrath meant for his people, or did he merely mute original sin and leave the destiny of our eternal souls in our own hands?
How we answer these questions has everything to do with what we think about our sin and the glory of Jesus, and therefore, it gets at the heart of the gospel.
What makes the gospel good news? Is it only the potential to be saved, which would grossly miscalculate human ability and undermine the blood of Jesus, or is it the declaration, “It is finished!” — that Jesus has demolished every obstacle that keeps his people from everlasting joy in God?
The Bible is clear. The gospel is God’s work, God’s victory. “He does everything, first to last, that is involved in bringing man from death in sin to life in glory: he plans, achieves and communicates redemption, calls and keeps, justifies, sanctifies, glorifies” (Packer, Quest for Godliness, 130). And here is where we see his love — the kind that breaks through the sternest soul in sovereign power to save. The love of Jesus is sin-crushing, serpent-stomping, death-defying, people-purchasing love.
Softly and tenderly tip-toeing around the doors of our heart, giving his life to give us a chance, crossing his fingers that we will invite him into our lives . . .
no, Jesus doesn’t love us like that.
Related resources:
From Heaven He Came and Sought Her: Definite Atonement in Historical, Biblical, Theological, and Pastoral Perspective (November 30, 2013)
Limited Atonement (“What We Believe About the Five Points of Calvinism”)
Four Keys to Satisfying Your Starving Soul [Desiring God Blog] 2013-10-19 01:00

His divine power has granted to us all things that pertain to life and godliness, through the knowledge of him who called us to his own glory and excellence, by which he has granted to us his precious and very great promises, so that through them you may become partakers of the divine nature, having escaped from the corruption that is in the world because of sinful desire. –2 Peter 1:3–4
If we’re honest, we’re all hungry. We’re starving for something to sustain us, to preserve our hope, to strengthen us through trials, to help us conquer sin. We’re starving for food that will fill us for the everyday fight of faith.
But what does the fight look like? And how do we find the food we need?
Our aim is to be with God and like God, to “become partakers of the divine nature” (1:4). Peter wants us to enjoy greater and greater fellowship and intimacy with God by becoming like him, by growing in godliness. We enjoy more life by being more like God, who is Life.
If we’re not careful, we easily slip into other aims that will rob us of life, aims that promise much and ultimately deliver very little — selfish gain, lustful thoughts, godless obsessions, excessive consumption, restless laziness. These aims may be easy and temporarily pleasing, but they only leave us hungrier. What our souls need is God.
If you’re believing in Jesus, forgiven and rescued from your sin, you are and will be still imperfect and broken. Our new aim in this new life is not perfection, as if that could earn us a place in heaven. Our aim is to live lives that are more and more pleasing to the Lord we love, and, by doing so, to experience more and more life and joy in him.
So if that’s our aim, what’s in our way? In order to be like God, we must escape “the corruption of sinful desire” (1:4). Our greatest obstacle to enjoying more of God is our own corrupt desires. They’re striving to starve our hungry souls and leave us begging for scraps along the highway of eternity. God knows better, and he offers us better.
The reality is I will suffer in this life, people will sin against me, and the devil lies in secret plotting to steal my hope and faith. But my greatest adversary is not suffering, sinners, or Satan. It’s me –– the lingering sin yet in my heart.
If we want to know God, be like him, be with him, we must be continually rescued from our sin in this life. For lovers of Jesus, this war has already been decided, and we’re now working out our victory every day until Jesus returns and ends the war once for all.
Jesus did the decisive work once for all on the cross, but we have a role to play. We have real choices to make. We must take steps to confront this enemy within us and be killing him.
The death of my sin sounds really sweet, until of course I try to kill it. Our greatest adversary, sin, is also our greatest handicap. It’s perfectly positioned to undermine the great aim of our new life. Praise God he doesn’t stake the battle on our ability. “His divine power has granted to us all things that pertain to life and godliness…”
The way to enjoy more of God is to live like him. And the power to live like him is not yours, but his. We find sure help in God’s hands, in his power — the power that formed mountains, that dug rivers, that lights stars, and breathes life into bears, sharks, and bald eagles; the power that establishes the universe, governs the nations, and judges all people. When you live by that power, you lack nothing on the road to godliness.
God “has granted to us his precious and very great promises, so that through them you may become partakers of the divine nature” (1:4). The ammunition for our daily battles are God’s promises — specific, blood-bought promises. This is the feast. When your hungry soul groans, this is what it wants. These promises are specific. You can find them, understand them, memorize them, and share them.
When we’re physically hungry, we don’t just talk about what food is. We find real food — a turkey sandwich on wheat, a Wendy’s cheeseburger with chili, a grilled chicken salad, some trail mix, or a Cliff bar. The idea of food does nothing for our hunger if we don’t identity something specific and edible and put it in our mouth.
The same applies to God’s promises. We don’t win battles over sin, suffering, and Satan by just acknowledging we need promises. No! What are they? How do they keep me from sinning or despairing or doubting? If promises are going to fulfill their God-given purpose, we have to know them, rehearse them, and voice them to one another. Promises like these:
We all are being transformed into the image [of Christ]. (2 Corinthians 3:18)
[God] will wipe away every tear from their eyes, and death shall be no more, neither shall there be mourning, nor crying, nor pain anymore. (Revelation 21:4)
He who began a good work in you will bring it to completion at the day of Jesus Christ. (Philippians 1:6)
At least as often as your stomach gets hungry, your heart and soul get hungry. Look for God’s promises when you read the Bible, specific promises, and feed your hungry soul. Eat them. Eat them every day and throughout the day. Eat full meals. Eat snacks. Eat planned meals. Eat spontaneously.
And as you do, you will become more like God. And as you become more like him, you will experience more of the abundant life he has given you and less of the sin from which he has rescued you.
Why Did God Forbid One Tree in Eden? [Desiring God Blog] 2013-10-18 11:42

Of the many trees in the Garden, God banned Adam and Eve from eating from one — just one (Genesis 2:16–17, 3:1–3, 11). Why?
John Piper recently gave the question some fresh thinking, which he shares in today’s episode of Ask Pastor John:
The function of the tree of the knowledge of good and evil is to make sure that the pleasures of all the other trees in the garden are supremely pleasures in God.
The command went like this: “And the LORD God commanded the man, saying, ‘You may surely eat of every tree of the garden, but of the tree of the knowledge of good and evil you shall not eat, for in the day that you eat of it you shall surely die’” (Genesis 2:16–17).
So what was God saying in prohibiting the eating of one tree out of a million trees? He was saying, “I have given you life. I have given you a world full of pleasure, pleasures of taste and sight and sound and smell and feel and nourishment. Only one tree is forbidden to you. And the point of that prohibition is to preserve the pleasures of the world, because if you eat of that one you will be saying to me, ‘I’m smarter than you. I am more authoritative that you. I am wiser than you are. I think I can care for myself better than you care for me. You are not a very good Father. And so I am going to reject you.’ So don’t eat from the tree, because you will be rejecting me and all my good gifts and all my wisdom and all my care. Instead, keep on submitting to my will. Keep on affirming my wisdom. Keep on being thankful for my generosity. Keep on trusting me as a Father and keep on eating these trees as a way of enjoying me. There are 10,000 trees, every imaginable fruit. Just go eat. Be thankful. I have given them to you and see them as expressions of my goodness and savor them that way.”
And Satan comes along, and he takes that arrangement and says, “Hey, Eve, the meaning of that arrangement is: God is selfish. God is stingy. He is a skinflint.” So he took the prohibition of one suicidal tree and treated it as a prohibition of everything.
So the issue of the tree is this: Will we keep looking to God as the giver and lover and treasure of this garden so that all our eating is thanking and all our savoring is a savoring of God? Will we keep on experiencing every one of these tastes as a tasting of something like what God is, and in that sense a tasting of God? Will we keep on enjoying God in the enjoying of the trees?
That is what the forbidden tree was there to test.
I think a lot of people try to set that up as merely arbitrary: Will man obey? Or will he not obey? And they don’t put it in the context of his fatherly care and all the goods that he has given. I don’t think it is arbitrary like that.
It was a warning. “If you choose independence instead of God-dependence, you will lose the pleasure of the garden and God with it.”
“If you keep trusting me and enjoying me as your greatest delight and highest treasure, you will have this garden and I will be the pleasure of all your pleasures.”
The forbidding one tree is a way of securing that the pleasures of all the other trees in the garden are supremely pleasures in God.
You can listen to the full episode here:
And here are three more recent popular episodes:
Ask Pastor John is a daily podcast series of 3–8 minute conversations released each weekday at 10:30am (EST) through the DG Facebook and Twitter feeds. You can tune in to the new episodes through the free Ask Pastor John mobile app for iPhone and Android. We’re currently hosting all the recordings on SoundCloud, a website making it easy to listen to several of the podcasts in one sitting. They’re also archived on the DG website and syndicated in iTunes.
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The Powerful Glory of Yielding Power [Desiring God Blog] 2013-10-18 01:00

Perhaps the only thing harder for prideful humans than humbly wielding power is humbly yielding power.
And the most beautiful Old Testament example of this is the way Jonathan yielded Israel’s throne to David. But as we see in 1 Samuel 23:15–18, he did far more than just yield.
Abinadab had watched his fugitive younger brother receive Jonathan like royalty. Such an embrace. Such intimate talk. Such weeping in farewell. What had David divulged to the enemy’s son?
He stepped beside David at the cave’s entrance and they watched Jonathan depart — returning to serve beside his father whose homicidal paranoia was forcing them to run like foxes and live like badgers.
“David, you won’t like my asking, but I need to. Is it wise just letting him go back to Saul?”
“My life is never safer than when it’s in his keeping.”
Abinadab shifted uneasily. “I know you love him. You’re very loyal. Very trusting. It’s one of your great qualities. I just hope your loyalty isn’t naïve here.”
David said nothing, his eyes still fixed on Jonathan.
Abinidab continued, “Brother, these are treacherous days. You barely escaped Doeg’s loose tongue. And those cowards of Keilah would have offered you as a peace offering to Saul despite the fact that you had just saved their necks from the Philistines. We need clear thinking here. Jonathan is next in line to be king. You’ve been friends. But the fact is, you’re now his one rival to the throne. Isn’t it possible that the blood of royal power may be thicker for Jonathan than the water of your friendship?”
Jonathan’s silhouette melded into the dusky shadows of the Horesh hills. David wiped his eyes and turned back into the cave. “You don’t know him, Abinadab. I’ll forgive this offense to his honor. We’re no more in danger than if that was our father walking away. But it’s not Jonathan’s affection for me that I trust. It’s his faith.”
Abinadab followed David. “Well, I hope I’m wrong, I really do. But Jonathan’s coming out here makes no sense to me if it’s not to spy you out. If he wanted to protect you he should never have come at all! What if he was followed?”
“Nobody’s more skillful at traceless trekking than Jonathan.”
“Maybe. But why would he come just for a friendly visit? Think of the risk. If his father finds out that he’s been here and didn’t report it, his life won’t be worth a pigeon’s. The king has nearly murdered him twice already! If he came here for love then he risked his life and all of ours. Why?”
“To strengthen my hand in God, Abinadab. Because he knows me. He knows how discouraged I can get.” David looked down and smiled. “God sent him because he knows how dark it’s been for me. I know what God has promised me. But with barely a step between me and death, it’s like I forget.”
David sat down on the rock near his gear and pulled some parchment from his satchel. “I’ve been working on this psalm. Let me read you the first lines:
My God, my God, why have you forsaken me?
Why are you so far from saving me, from the words of my groaning?
O my God, I cry by day, but you do not answer,
And by night, but I find no rest. (Psalm 22:1–2)
“Today,” David paused, clenching back sobs. “Today Jonathan risked his life to help me rest—to remind me that God is not far at all. What he said to me was, ‘Do not fear, for the hand of Saul my father shall not find you. You shall be king over Israel,’” David paused again as tears flowed freely, “‘and I shall be next to you. Saul my father also knows this.’
“Jonathan believes God, Abinadab. It’s his faith I trust. Jonathan loves God more than he loves power. And more than he loves me. He loves me because he loves God. That makes him the safest man in the world to me. He has no equal.” David hung his head. “I only hope he survives his father’s insane faithlessness. I so desperately want him next to me.”
David had a very difficult calling: to wield the power of Israel’s kingship with God-dependent humility.
Jonathan’s calling may have been more difficult: to yield the power of Israel’s kingship with God-dependent humility.
But Jonathan didn’t just yield to David. He loved David (1 Samuel 18:1), empowered David (1 Samuel 18:4), protected and advocated for David (1 Samuel 20). And when David’s faith-hand was losing its grip, he sought him out and “strengthened his hand” by reminding him of God’s promises (1 Samuel 23:17). He could have only done this if he trusted in the Lord with all his heart (Proverbs 3:5).
Like Jonathan, God wants us to seek first the kingdom (Matthew 6:33), not our prominence in it. When we trust God enough to yield our prominence (or expected prominence) to someone else for God’s purposes it’s a sign and wonder. And when we go beyond that to doing everything in our power to help them succeed — nothing else quite images the Philippians 2:5–11 glory of Jesus.
Jonathan did not consider the throne a thing to be grasped, but he made himself nothing for God’s sake and became a Christ-like servant. Let us also “have this mind” (Philippians 2:5).
This story is from 1 Samuel 16:8; 1 Samuel 22:9–19; 1 Samuel 23:1–14; 1 Samuel 14:24–46, 20:30–34; and 1 Samuel 20:3.
Lecrae Unwraps His Joy in Jesus [Desiring God Blog] 2013-10-17 11:30
How do you pursue joy in Jesus? I mean what does it really look like — like today? How about when things are crazy at work or tense at home? If God made us to enjoy him, we have something infinitely valuable at our fingertips at any given moment. But how do we tap into it?
In this two-and-a-half-minute video, Lecrae Moore talks about his joy in Jesus — where he finds it and how it works itself out in every arena of his life. For him, fighting depression and discouragement is about spending lots and lots of intentional time with Jesus. As in every other relationship, Lecrae says, “quantity brings about quality,” because eventually we will see Christ more clearly.
Related resources:
Cooking for Eternity: The Story of Jeff Ansorge [Desiring God Blog] 2013-10-17 01:20

ST. PAUL, MN — Jeff Ansorge is a trim 40-year-old with buzzed hair and sideburns that are mostly silver and thick eyebrows that are mostly black. He’s a quiet, t-shirted Midwestern guy who can be found on any weekday hard at work in the Salvation Army soup kitchen in northeast St. Paul, Minnesota.
Every weekday morning, Jeff fills an echoing, cinder-block gymnasium with folding chairs and utility tables as he runs the lunch preparations in the kitchen off to the side. Before the day is done, he (and a volunteer or two) will serve between 140 and 180 lunches to a single-file line of the poorest residents in the community.
After twelve months, Jeff’s work is becoming routine. Managing the menu, ordering food, unloading pallets of food, coordinating his volunteers — all part of his daily juggle of set up, serving, cleaning, wiping down tables, stacking chairs, cleaning the kitchen, and taking out the trash when the lunch session is over.
It’s a daily routine Jeff enjoys, which is remarkable for a man who was, until recently, one of the highest paid chefs in the Twin Cities.
Jeff was born in rural Minnesota and raised in rural Wisconsin by a devout Roman Catholic family. For much of his boyhood he served six days a week in his church and dreamed of becoming a priest.
Raised on a steady diet of Midwestern casseroles, his culinary interests were first triggered by a family trip to Disney World where he encountered a five-course French meal. The experience was so profound it sparked in him an inextinguishable new desire for the world of culinary art. Jeff began cooking. He cooked for the family, he cooked in home economics class in high school, and he watched the cooks in the local restaurant business while working as a busboy and dishwasher.
In high school Jeff entered and won a statewide recipe contest sponsored by one of the top culinary schools in America — Johnson & Wales University in Providence, Rhode Island. His recipe (a take on ocean perch), earned him a trip to compete in the national competition at Johnson & Wales. He won scholarships and decided to attend the school, 1,200 miles away from home.
Jeff excelled in college, and his success fueled his passion for cooking. The freedom away from home was liberating, and it opened him to all the blessings and all the temptations of college life. After his first year, Jeff fell into vices that would plague him until his early 30s — drunkenness, addictions, and drugs.
Despite his vices, Jeff continued to excel in his culinary career path. In part, he supported himself through four years of college by working, first at a local Subway near campus, then later by teaching at the school. After college he became a prep cook with The Capital Grille, an upscale steakhouse on the rise in Providence. The timing was perfect. As Jeff worked up the ranks to sous-chef the company began expanding to its over forty locations around the country. Jeff was tasked to open new restaurants.
Jeff returned to the Midwest in 1997 when he opened The Capital Grille in downtown Minneapolis, a five-star, New York style chophouse and sophisticated men’s club, with an ambiance of mahogany, leather, chandeliers, and thousands of wine bottles.
It was there he met his wife, a hostess. They were married in 2002 and had their first child in 2005.
The restaurant life was exciting, but by the time he became a father in 2005, Jeff was burned out from the demanding schedule of an executive chef. He left the kitchen, though not for long. After leaving The Capital Grille he eventually made his way back to the kitchen at a jazz club steakhouse in downtown Minneapolis. But when the Capital Grille lost their head chef, they enticed him back with six-digits’ worth of salary, bonuses, and stocks.
He made the move, but as the months and years passed, a deep void in his life became increasingly visible to his wife and his family. He was depressed, and began to rely on antidepressants. He no longer smoked pot or cigarettes after his first child was born in 2005, but he drank sporadically at night after work. His marriage grew increasingly unstable, and though he tried to fill the void in his life with work, money, alcohol, and pornography, the emptiness grew. A breaking point was near.
Late in 2010, Jeff’s life really crumbled.
“I crashed with my depression. I wanted a divorce. In the fall of 2010 two of my brother-in-laws confronted me. ‘You cannot do this,’ they said. They came at me from a biblical standpoint. They would confront me with Scripture over the phone, then I would hang up and my wife and I would laugh and talk about how crazy they were.
“One day they traveled from Iowa and Milwaukee, sat me down in my kitchen, and asked me about basic Christian truths: Do I believe in Jesus? Do I believe that God created the world and Adam and Eve? And I said, yes, absolutely. They both told me about their lives and explained spiritual warfare we face in this world. The next morning in my car I turned on the AM radio station and heard a preacher say word for word the same thing my brother-in-law said the night before. That’s when it happened — God opened my eyes.”
Jeff always thought of himself as religious, but he didn’t talk about it. Faith wasn’t a hang-up when he married his wife (an agnostic).
But now he had experienced the regenerating grace of God in his life. Never a reader, Jeff got a copy of the Bible and read the New Testament for the first time. He read it in ten days. His new zeal was becoming obvious, but his newborn life only drove a wedge deeper into his marriage. One month later, his wife asked him to move out, filed divorce papers, and ended their nine-year marriage.
As the months passed in his new life in Christ, Jeff found himself less and less interested in maintaining the lifestyle at The Capital Grille. The company had grown into a mammoth corporation and Jeff was ready to move on, permanently.
Having experienced God’s grace, Jeff turned to the non-profit food industry where he could integrate his love of food and his burgeoning desire to share the gospel. He submitted his résumé to the Salvation Army and several other non-profit organizations. After a few long months of silence and waiting, he was contacted, interviewed, and hired.
Jeff left a restaurant that averages a bill of $80 per person to serve lunches at a cost of $0.63. A lot of people thought he was crazy to make the move. But even making $100,000 a year, Jeff didn’t own his house or cars — he couldn’t escape his debts. When he left The Capital Grille, he decided to sell off his employee stocks to pay off his cars and his house and all his debts.
“Now I make less than 40% of what I once did. I’m on the clock. I get paid by the hour. But I’m out of debt. I own my house and cars. It was worth it.”
So what’s an average day for Jeff now?
He runs the hot lunch program, and during the school year he cooks for an at-risk youth program (snack and dinner). “An average day for me includes menu planning, ordering food, sorting food donations, putting bills into spreadsheets, cooking, organizing and managing volunteers and community service workers, and then offering a biblical devotion and meals to the clients who come through. I help with Friday food giveaways; we move 3,000–5,000 pounds of food for people to have with them over the weekend. I mop, I clean, I set up tables, I set up chairs, I take out the garbage. I’m the only paid person here.”
Nothing is below his pay grade — sometimes, when volunteers don’t show, he pulls off lunches by himself.
Jeff’s dramatic transition from a five-star kitchen to a Salvation Army gym raises two questions.
First, why not remain in the high-end food industry and be a witness for Christ there? “In the restaurant industry I cannot go out in the dining room, stand there and preach the gospel to everyone,” he said. “But now I can. I feel better utilized among people who are homeless or have low incomes, and feel I have a greater good to offer here.”
But there’s a second question. Given his Roman Catholic background, is all this non-profit work his penance to make up for a previous life he regrets?
“Not at all,” he says without hesitation. “That never entered my mind. When I was a kid and I would do something wrong, I would confess, and the priest would assign seven ‘Hail Marys,’ or whatever. I know what penance is, but this is not penance. I’m doing this because I was reborn, because something tangible happened inside of me because of the gospel, something that I can define and feel and pinpoint the time and events in my life surrounding it.”
The joy on his face proved the point.
This soup-kitchen cook serves out of a profound identity transformation. “My identity was determined by my job: by my status, the money I was making, and the house and cars I could buy. Now, my identity is in Christ: I am a follower of Christ, a child of God. My identity is now someone who seeks to help people and to spread the gospel. I’m doing what I love, for people I love, for the person I love — Jesus Christ.”
After a year, Jeff is starting to see a return on investment in the lives he serves. “I’ve seen so many people come through the door that are hard. When I first got here there was one guy in particular, I would ask his name and he would tell me, ‘I just came here for a meal, that’s all.’ And that was it. He ate and left. Months later now this same guy comes in and we chat and engage in conversation. I have seen this tangible life change, knowing it happened by being a reflection of Jesus.”
Jeff has been changed this year, too. “I came from working in restaurants all my life. Very driven. High speed. All about organization, and making things as streamlined and efficient as possible. It took me six or seven months to wind down from that. When I got here I was all about streamlining, efficiency, changing things, making it good — so focused on the food. At the same time I was leading the devotional. But over time God has opened my eyes to see the food is secondary.”
Except for the man who took his application and hired him, Jeff’s résumé was a secret when he started at the Salvation Army (his preference). But his story soon got out, spread, and was made public when a local television station featured him. His volunteers now call him “Chef Jeff.”
After his first year, he is happy, and says the move to The Salvation Army was one of his best life decisions.
It’s a story filled with sin and grace, and it’s a story Jeff is willing to share, if you ask him. But he’s not eager to share his private or public story. “I’d rather be tucked away,” he says in his tucked-away office. He says he looks forward to a day when the reporters and the spotlight leave him alone, when he is no longer given stewardship and community awards, when he appears on no more Salvation Army posters, or in magazine articles, or in blog posts, when he can quietly continue the mission he has taken up to the poor who show up every day to his gym-turned-dining room, to fill their hungry stomachs with food that perishes, and — more importantly — to offer hungry souls the all-satisfying bread of Jesus Christ that endures for all eternity (John 6:27, 35).

More biographical profiles from Tony Reinke:
The Remarkable Reality of Union with Christ [Desiring God Blog] 2013-10-16 11:33

It may be the most important doctrine you’ve never heard of.
So says Kevin DeYoung about the reality of “union with Christ” — and sadly it seems he’s right.
But hopefully you aren’t among that number — and have not just heard about the doctrine, but begun to taste the practical fruit of this wide and wonderful reality. As Sinclair Ferguson says about union with Christ, “Of all the doctrines surrounding the Christian life this, one of the profoundest, is also one of the most practical in its effects.”
Even though union with Christ too often has been under-emphasized, more and more pastors and laymen are talking in recent years about union with Christ. And we want to add kindling to that fire.
Simply put, union with Christ is the theological term for the believer’s being joined to Jesus by the Holy Spirit through faith. In fulfillment of God’s ancient promise, I will be your God, and you will be my people, union with Christ is, according to John Frame, “the most general way of characterizing Jesus’s work of salvation.” The way in which all that Jesus accomplished for us gets applied to us is through our being connected to Jesus, our being “in” him.
Our union with Jesus by faith is how the objective accomplishments of his life, death, and resurrection two millennia ago come to be subjectively applied to other humans, whether Peter, Paul, Mary, Augustine, Luther, Spurgeon, or us who believe in the 21st century.
Two voices who have helped get this vital doctrine back on the radar, and into its proper place, are Sinclair Ferguson and Michael Horton. We dreamed that maybe they’d help us explore the glories of this doctrine at our upcoming Desiring God 2014 Conference for Pastors, and they both agreed. So Monday to Wednesday, February 3–5, 2014, Ferguson and Horton will join forces with John Piper under the banner,
The Pastor, the Vine, and the Branches: The Remarkable Reality of Union with Christ
We’d love to have you mark your calendar, make arrangements, and join us in Minneapolis in February. Ferguson, Horton, and Piper each will give two plenary messages on union with Christ — Ferguson focusing on exposition, Horton honing in on the theological and historical, and Piper preaching through John 15 and providing a biographical address on missionary Hudson Taylor.
A “early bird” registration rate is available for those who get in early, by Monday, November 11.
Also, Paul Tripp will be joining us for two pre-conference sessions on making the gospel practical in pastoral ministry, along with four afternoon seminar options. Here’s the full schedule, with the speakers and message titles.
1:00 – 1:45 PM Pre-Conference
2:00 – 2:45 PM Pre-Conference
3:00 – 3:45 PM Seminars
Jared Wilson, The Promise in Pastoral Weakness
Kempton Turner, The Pastor’s Purity and Pleasure
Rick Gamache, Pastoring Your Wife and Kids
David Mathis & Jonathan Parnell, How to Stay Christian in Seminary — And Any Season of Waiting
7:00 – 8:30 PM
John Piper, Glorifying God by Bearing Fruit in Union With Christ (John 15:1–11)
8:30 – 10:00 AM
Sinclair Ferguson, Union with Christ: Mind-Renewing Foundations
10:30 – 11:30 AM
Michael Horton, Calvin on Union with Christ
1:45 – 3:00 PM
Sinclair Ferguson, Union with Christ: Life-Transforming Implications
7:00 – 8:30 PM
Michael Horton, Union With Christ and the Communion of Saints
8:30 – 10:15 AM
John Piper, The Ministry of Hudson Taylor As Life in Christ
10:45 – 11:45 AM
Speaker Panel, Questions & Answers with Horton, Ferguson, and Piper
For more on union with Christ, especially as it relates to sanctification, see the introduction to Acting the Miracle: God’s Work and Ours in the Miracle of Sanctification and John Piper’s post “The Stupendous Reality of Union with Christ”. For more information on the conference — or to take advantage of the “early bird” rate by Monday, November 11 — see the event page.
The Subtle Art of Destroying Humans [Desiring God Blog] 2013-10-16 06:09

My dearest Grubnat,
I am glad to see you are finally learning to be subtler in manipulating your human. As I had warned you, I was concerned that your boisterous assault on the unborn vermin with the rare chromosomal makeup (the “disabled,” as the other vermin call them) was going to expose all our plans to destroy them.
So I congratulate you on the recent article in The New York Times, “Breakthroughs in Prenatal Screening.” I can see your skills developing. We must continue on this path as it does two important things for us: 1) it further blinds the humans to our real schemes; and 2) it rids us of having to deal with those foul, weak, “special” children that the Enemy calls “indispensable.” We mustn’t lose our grip here.
Because of this article, and unlike my last letters to you, I actually have a few things to commend — things to see you repeat.
Now, I wouldn’t have opened the article exactly the way you did (more on that in my comments about how you can improve), but I must admit I appreciate how you dehumanized the mother and the child in your opening paragraphs.
Refusing to mention the name of the child was excellent! Names are bad for our cause. It makes the humans actually begin to imagine them as “children” rather than the “mere tissue” we want them to think those little humans are. Let them only think the child is “Down syndrome,” and never Michael or Elizabeth.
Also, good job on not quoting the mother at all about how she feels today about her child. Those puny humans, left with all their preconceived notions and prejudices, already do well at creating frightening alternative realities for that mother. They talk about how sad she was, how horrible her life was, how much better it would have been if that child had never been born. In fact, I am a little jealous at how quickly they’ve been trained to distort reality.
The humans who have been through the distortion tutelage usually aren’t even afraid of the right things! When a parent talks about both the hardship and the “blessing” of raising that child (yes, the Enemy often makes good come to the family through this vermin), a little whiff of reality sets in. And when that happens it becomes harder for us to kill the next one. So keep bleaching from their minds the idea of these babies being real persons.
Another thing: the clean, clinical description of the prenatal tests nicely ignored any discussion about how much the culture loathes these disabled children. How laughable for them to think that “tests” are neutral in a world like theirs!
The doctors and so-called ethicists are the best among them for our cause. Keep working on the medical professionals to believe that termination is a responsible choice, especially when you know the mother doesn’t have anyone around her to support the birth of her child.
And another thing: The following sentence was perhaps your best: “When prenatal testing reveals that a fetus has a serious birth defect, some women may consider ending their pregnancies.” Hilarious! Yes, yes, yes! Let the mothers think they are just making an “informed, personal choice” rather than realize that they’re part of our culturally orchestrated attack on children who have the extra chromosome. The Holocaust was fun, but this is even more satisfying. They actually think their individual choices don’t accumulate into genocide.
Now enough with the commendations! Here are a few things you neglected.
Why in the world did you allow them to have drawings of babies in the graphic above the article? Yes, some humans are so calloused that they don’t care whether it is a baby or not. But most don’t like to think too much about it. Don’t give them any evidence that the little creatures they are destroying are actually human just like them. Don’t make this mistake again. Test tubes or computer code or something like that are far better. Keep it clean and scientific.
As much as I appreciated your opening the article with the story about the friend with the disabled child, you overdid it. The headline was about prenatal screening and you immediately made the story about abortion. The humans are stupid but even they can see an agenda this obvious. Next time bury the abortion bit later in the story so that the test results always end with a “compassionate” termination.
Never suggest that they talk to parents who are raising children with disabilities. For some unknown reason to me, when they talk to people who have experienced real suffering rather than imagined suffering, they get encouraged and emboldened. You know how our Enemy works in suffering.
In the future, only present all the hardship and expense and loneliness and how awful their lives will be, always couching it in terms of how horrible their baby’s life will be. They feel better, and their hearts get just a little harder, when they rationalize that killing their baby was a good thing. That’s the reason that if we can convince a woman to have an abortion she will likely have more.
Make these corrections and I’m sure your next piece will be even better. And I know The New York Times will be happy to print it.
Content with your work, for now,
Wormwood
Recent posts from John Knight:
I Hate Porn [Desiring God Blog] 2013-10-15 10:56

Pornography is a problem.
Porn is like a narcotic, it hijacks the brain, it redefines human sexuality, and in the meantime ruins lives, destroys families, and destabilizes ministries. And honestly it’s a problem that makes me tired — tired of the devastation Satan is causing to children, women, families, pastors, churches, and the world with this tragic evil.
Porn became a problem for me when I was only six, and by the grace of God that problem ended when Jesus saved me at age seventeen. But I know it rarely happens so cleanly. It is still a temptation, yes; temptation abounds living in the city I do, and with the heart I have, but grace abounds all the more in Jesus Christ.
Friends, I hate porn. And here’s why.
I hate porn because it is a perversion of what God created in man and woman.
I hate porn because it exploits women made in the image of God into an image made for a man’s lust.
I hate porn because it objectifies women into a consumable product instead of a glorious image-bearing creature of God.
I hate porn because I love women — in particular my wife and three daughters.
I hate porn because it takes the soul satisfying experience of sex with a covenantally-committed spouse and turns it into a twisted soul shrinking experience of self-sex.
I hate porn because it turns sons and daughters of God into slaves of sex.
I hate porn because it turns potential missionaries into impotent Christians.
I hate porn because it destroys marriage, many before they even begin.
I hate porn because it extends adolescence and keeps men boys.
I hate porn because it lies to men about beauty and leads men to look for a porn star instead of a woman who fears the Lord.
I hate porn because it robs men and women of the full joy of obedience.
I hate porn because it fractures trust between a husband and wife.
I hate porn because it is a diabolical, satanic activity that is subtly leading thousands upon thousands to hell.
I hate porn because it leads to disqualified pastors and impotent churches. (Pastors, if you are addicted to porn, you are disqualified, and you are killing your church!)
I hate porn because I suspect it’s the most significant reason we are not planting more churches and sending more missionaries.
I hate porn because it disqualifies gospel preachers who could fill the empty church buildings in my city and so many others.
I hate porn because of the disappointment children have to go through when their dad tells them why they lost their job or opportunity to lead in the church.
I hate porn because it teaches a distorted view of sex to children before it can be explained by loving parents.
I hate porn because I am tired of sitting in my living room with sobbing, confused, devastated wives and broken, embarrassed, condemned men who got caught.
I hate porn because it leads to rape, molestation, and perversion that can devastate people for the rest of their lives.
I hate porn because it turns men inward and suffocates a man’s ambition to make God’s name hallowed.
I hate porn because it says sin, Satan, and the world are more satisfying than our Triune God and his grace.
I hate porn because I hate ungodly guilt and condemnation.
I hate porn for the fear it induces in the hearts of parents everywhere that their child could stumble upon a sight and get addicted.
But I love Jesus.
I love Jesus because he loves people with porn problems.
I love Jesus because he is powerful to free porn-enslaved hearts.
He who knew no porn addiction became porn addiction so the porn addict might become the righteousness of God in him.
He who had no sin became sin for you so that you may become the righteousness of God (2 Corinthians 5:21).
In that one brilliant sentence, Paul puts an end to the porn problem.
Friend, you are no longer in Adam but in Jesus. Jesus became a substitute. It was as if he became the porn addict, by receiving the just penalty due for our perversion, and you became the righteous son or daughter of God with all its benefits.
Friend, in one act of Love and Justice, in the cross-work of Jesus, through faith in him, you are now clean, holy, accepted, forgiven and free. Let me say it again . . . free!
I love Jesus.
Recent, related posts:
Pornography: The New Narcotic (John Piper)
Hijacking Back Your Brain from Porn (John Piper)
Porn, Pride, and Praise (Heath Lambert)
Hijacking Back Your Brain from Porn [Desiring God Blog] 2013-10-15 01:00

Last week I wrote about the physiological dimension of addiction to pornography. New brain research suggests it is as strong as addiction to cocaine and heroin because of its unique combination of stimulant and opiate. Pornography lays down real physiological paths in the brain. All sexual experience tends to migrate to these paths.
I concluded that none of this brain research takes God by surprise. He designed the interplay between the brain and the soul. Discoveries of the connections between physical and spiritual reality do not nullify either.
So don’t let this new brain research make you think of yourself as mere flesh and chemicals. This is the great myth of the modern world — what C. S. Lewis called the abolition of man. This is the theory that human thought is nothing but movement in the brain. It is a theory developed to destroy itself.
Lewis saw the tentacles of materialism reaching into every sphere:
There will always be evidence, and every month fresh evidence, to show that religion is only psychological, justice only self-protection, politics only economics, love only lust, and thought itself only cerebral biochemistry. (“Transposition,” in The Weight of Glory, 114–115)
But Lewis saw that nobody really acts as though they believe this. They are playing a language game. He illustrates this with the relationship between thought and brains:
We are certain that, in this life at any rate, thought is intimately connected with the brain. The theory that thought therefore is merely a movement in the brain is, in my opinion, nonsense; for if so, that theory itself would be merely a movement, an event among atoms, which may have speed and direction but of which it would be meaningless to use the words “true” or ”false.” (“Transposition,” 103)
Lewis is not playing counter-games here. He is blood-earnest that the abolishers of man are refusing to see that they claim to make meaningful statements while destroying meaning.
Meaning is rooted in supra-material truth. You are not mere matter and energy. You are an embodied soul who will live forever in heaven or in hell, created in the image of God, unlike mere animals, and, as a Christian, bought with the blood of the Son of God, and indwelt by the very Spirit of God himself. These are stupendous realities — greater realities than endorphins and dopamine.
God wove together physical nerves and supra-physical spiritual affections — desire, fear, joy, anger, pity, admiration, trust, cherishing, love. Instead of letting this connection discourage you, take it by the horns and make it serve your holiness. This is what the Bible calls you to do.
Don’t think the Bible is silent on this all-important question of mind and body — thinking and brains, affections and chemicals. God made these connections between physical and supra-physical, and God has wisdom for living in them.
Consider these four hope-filled observations.
Brain research is an infant science, publishing its first baby steps. They have scarcely begun to even name the mysteries of how truth and beauty is mediated through language, and then enters the mind as thought, and then is transposed into corresponding chemical processes.
Therefore, we should take hold of this amazing connection and claim what the Bible claims: Beholding the glory of the Lord, we are being changed (2 Corinthians 3:18). Of course seeing nudes changes the brain. But why should we think that seeing the glory of Christ exerts a weaker change? If brain paths pervert our affections and our behavior, do not make the wild mistake of assuming sanctification can only make weaker paths.
Paul calls you to “be renewed in the spirit of your minds, and to put on the new man, created after the likeness of God in true righteousness and holiness” (Ephesians 4:23–24). Watch out, lest you assume that the renewal of “the spirit of the mind” leaves no trace in the paths of the brain. It does.
Paul says, “Put on the new man, which is being renewed in knowledge after the image of its creator” (Colossians 3:10). If the seeing of internet nakedness creates new paths in the brain, how much more the seeing of Christ — the spiritual sight of “the gospel of the glory of Christ who is the image of God” (2 Corinthians 4:4). We are not left to create new brains for ourselves: “we are his workmanship, created in Christ Jesus” (Ephesians 2:10). Do not be cowed by brain research. God made the brain and wrote the Book.
Moreover, we know from experience that we are not slaves of these powerful pornographic changes in our brains. I do not minimize them. Judging by the ongoing effects, even in my sixties, of my teenage tomfoolery, I have tasted the amazing staying power of old sinful patterns. But we are not horses or mules that can only be curbed with bit and bridle (Psalm 32:9).
You know this. If you were in the grip of great sexual desire for pornography, and Jesus himself stood before you in your room, blood splattered, hands trembling with pain, eyes brimming with love, breathing heavily like a dying man, you know — yes, you know — you would have power in that moment to not look at the pornography as Jesus stood there. You are not enslaved. The well-beaten neural paths in your brain would not win. They are not God. They do not have the last say.
Or just at the physical level, you know from experience that a mere smell — say of human feces, or rancid garbage, or your own armpit can knock the sexual drive right out of your groin. What does that mean? It means those neural paths are not final. They can be trumped. You are not a mere victim.
Or consider this. You are about to commit fornication in a tent in the woods. You never dreamed it would come to this, but now the tidal wave of desire has simply conquered you. Or has it? What if, in the moment of hottest passion, before entry, you heard the sound of a grizzly bear, and saw silhouetted on the tent his mammoth size, would you be the slave of lust? Or would not fear utterly triumph over those chemicals?
Beware of thinking you are a victim of the euphoric effect of dopamine and endorphins. You are not. God has ways of revealing his bloody Christ, and staggering you with odors and bears to rescue you for himself. He will stoop to this for love’s sake.
Supra-chemical emotions — spiritual affections — are transposed into corresponding physical responses in the brain. That means you can fight physical fire with spiritual fire. And it works the other way as well. God ordains that we fight for spiritual fruit by wielding physiological weapons with spiritual hands.
Have you ever considered the stunning implications of Paul’s Satan-defeating sexual counsel in 1 Corinthians 7:5? Be careful, single people. You are likely to jump to the conclusion that this is either irrelevant for you, or bad news. It’s not. Paul says to husbands and wives,
Do not deprive one another [of sexual relations], except perhaps by agreement for a limited time, that you may devote yourselves to prayer; but then come together again, so that Satan may not tempt you because of your lack of self-control.
This implies that Paul intends for Christian couples to fight against the supernatural power of Satan by having sufficiently frequent sexual relations. To put the point physiologically: There are brain chemicals that increase desire for sex as the length of abstinence increases. The power of those chemicals decreases after orgasm. Therefore, Paul says, make use of that physiological reality in marriage to reduce your vulnerability to Satan’s temptation to adultery and pornography.
Of course, this is not the only or the main weapon in our arsenal. But it is one. And it illustrates the validity of using physiological weapons against physiological foes. Single people may rightly say, I don’t have that particular marriage weapon in my arsenal. That’s right. And I admire you for saying it. But embrace the principle as it applies to you. There are physiological realities that you know affect your vulnerability to temptation. Use them to make war.
But is that spiritual? Isn’t self-control a “fruit of the Holy Spirit,” rather than a fruit of frequent sexual relations?
It is a fruit of the Spirit (Galatians 5:23), but not “rather than” a fruit of other forces. The Spirit’s way of producing his fruit often includes very natural means. For example, another fruit of the Spirit is patience (Galatians 5:22). But who of us would deny that our patience rises and falls with how much sleep we get? Love, Paul says, is “patient . . . and not easily irritated” (1 Corinthians 13:4–5). But we are more easily irritated, and less patient, when we have not gotten the rest we need.
What I infer from this is that one of the many weapons in the arsenal of the Holy Spirit is sleep. He humbles us to realize we are not God and that we need to be as helpless as a baby seven or eight hours a day, in order to be the loving, patient people he calls us to be.
Similarly with sexual self-control. The Holy Spirit teaches us from Scripture, and from experience, and from each other, how our bodies work. He means for us to lean on his power as we use the physiological counter-weapons he gives us.
Brain research is right: Our brains are deeply shaped by what we see. And the more we see, the more well-beaten and controlling those paths become. But we are not their victims. These physiological powers are not ultimate. God is ultimate. And he has given us spiritual weapons just as physiologically powerful as pornography. He too means to be seen — often and deeply (2 Corinthians 3:18; 4:4).
Moreover the spiritual powers of his word and Spirit have the right to conscript physiological forces into their service. And in the end, God can hijack back the very paths of pornography and transpose the scintillations of those very paths into the ecstasies of knowing Christ.
Recent posts from John Piper:
A Reason to Be Really Offended [Desiring God Blog] 2013-10-14 01:00

Our message is not you can do it.
It’s not you’re good enough, smart enough, and people like you.
What we preach is that you are a glorious creature gone tragically bad, that you have rebelled against the God who made you, but that he did the most difficult thing imaginable to win you back and lavish you with his eternal goodness.
It is wondrously good news. But unavoidable is the offense, that insulting supposition, that bad news that sets up the good. Did you catch it? You’ve gone tragically bad. You’re a foolhardy rebel against the most powerful person in the universe. There’s nothing you can do to save yourself, earn God’s favor, or get yourself out of the cosmic pit you’re in — the pit you dug and can’t climb out of.
The offense is that the magnitude of God’s solution — the slaughter of his own Son — shows the magnitude of our wickedness and frailty and utter inability. Yes, the gospel says you’re more loved than you ever could have dreamed, but as Jack Miller and Tim Keller have noted, at the same time it says you’re more sinful than you ever imagined. And that’s repugnant to the natural palate.
If you’ve never tasted the cross as offensive, you’ve missed something essential.
Talking about the cross as an “offense” comes from Galatians 5:11: “If I, brothers, still preach circumcision, why am I still being persecuted? In that case the offense of the cross has been removed.” Why it is that the cross would be seen as an offense? What’s offensive about the crucifixion?
The cross declares how dire is our condition apart from Jesus. It announces how deep the sin goes, how profound the rebellion is, how impossible is our plight apart from Help from the outside. There’s nothing we can do, no effort we can exert, no law we can follow.
The message of Christ crucified says you’re an absolute failure in relation to what’s most important. The horror of killing the Son of God points to the horror of our condition. The badness of Good Friday is a tribute to the badness in us.
The cross embodies some of the most offensive things possible you could say about someone in relation to God and eternity. This gruesome death Jesus died, you earned it. The hell Jesus endured, you deserved it, forever. The shame he underwent, the scorn, the disrespect, the hurt — all these are as suitable to us sinners as they’re unsuitable to the sinless one.
And it’s not that it just turned out this way, but God planned it. He designed the offense. Seven hundred years before Jesus, the prophet Isaiah called it — he will be “a stone of stumbling and a rock of offense” (Isaiah 8:14). And such he was, and continues to be. Both Peter and Paul pick up on the theme (Romans 9:32–33; 1 Peter 2:7–8).
Jesus himself, in John 6, challenges his disciples with the offense. “No one can come to me unless the Father who sent me draws him” (John 6:44). You can’t do it. You’re not good enough or smart enough. And perhaps most offensive of all: You are lifeless. “Unless you eat the flesh of the Son of Man and drink his blood, you have no life in you” (John 6:53).
The offense is not mainly his mention of eating flesh and drinking blood, but the accusation of deep depravity and spiritual inability. As the crowds retreat at his forthrightness, Jesus asks his disciples, “Do you take offense at this?” (John 6:61). More unnerving than taking his plainly figurative language in a literal sense is hearing that you are powerless and lifeless where it matters most. This is as offensive as it gets.
Typically we get antsy about speaking the gospel to someone who doesn’t already believe. Some of our fear, of course, is unwarranted. But some of it is for good reason. In communicating the gospel, one of the essential things we must at least imply, if not make explicit, is the most offensive truth possible: you are powerless precisely where it matters most. You are dead to what truly is life.
Don’t take it too far. We don’t gloat in giving offense. We labor to remove every possible barrier. May the offense not be our personality or carelessness or quirkiness or arrogance. Like Paul, let’s “endure anything rather than put an obstacle in the way of the gospel of Christ” (1 Corinthians 9:12; 2 Corinthians 6:3). Let’s strain to “become all things to all people, that by all means [we] might save some” (1 Corinthians 9:22). Let’s do everything in our power to “give no offense to Jews or to Greeks or to the church of God” (1 Corinthians 10:32).
But this one offense — the offense of the cross — we cannot remove. We dare not.
More from Sent:
The Best Way to Start Your Everyday [Desiring God Blog] 2013-10-13 01:00

Psalm 143:8 may capture it best:
Let me hear in the morning of your steadfast love,
for in you I trust.
Make me know the way I should go,
for to you I lift up my soul.
Two things make up everyone’s everyday: receptivity and productivity. We are creatures who constantly take in and put out. We absorb and we exude.
The question is what will it be?
The psalmist tells us the best way to start.
David writes as a man in turmoil. The enemy has pursued his soul (Psalm 143:3). His spirit is blacking out. His heart is appalled (Psalm 143:4). Don’t picture him waking up mid-morning at a cozy B&B. It’s more like a battle, and his enemy is in motion.
It’s more like how it is most days for us, even though the warfare against us is largely unseen. Our alarm clock goes off, and the prince of the air never hits his snooze. We wake up in a warzone.
Sometimes that may mean that we, by faith, cherish some extra time under the covers and stick it to our enemy. The decisive battle has been won, after all. Our blood was spilt for victory when our sins were nailed through Jesus’s hands. When he died, we died. When he conquered death, we conquered with him, and every force set against us was exposed to open shame. The hope of our eternal souls is not dependent upon how quickly we spring out of bed.
But we will get up, and whenever we do, the scheming principalities are soaring. Circumstances breathe down our necks. Stress starts pounding on our doors. We gear up for 17 straight hours of receptivity and productivity.
This is where David’s prayer in Psalm 143:8 helps. He gets to the heart of the matter. If we are stepping into absorbing and exuding, first things first, let us soak in God’s truth and then submit all our doing to his guidance. The best way to start the day, everyday, is with the prayer, in short: let me hear and lead me on.
Let Me Hear . . .
Let us first hear of God’s steadfast love and steady our thoughts on this anchor: God shows his love for me in that while I was still a sinner, Christ died for me (Romans 5:8). For my sake, God made his sinless Son to be sin so that in him I might become his righteousness (2 Corinthians 5:21). Christ redeemed me from the curse of the law by becoming a curse for me (Galatians 3:13). Let us receive his words: You did not choose me, but I chose you. I have redeemed you. I have called you by name. You are mine (John 15:16; Isaiah 43:1).
Rest firmly in the gospel of his grace — that he loves you, that he has his grip on you, that he is never letting you go (John 10:28). Let us hear this because it’s in God we trust, not the hottest news story, nor the latest best-selling book, nor the lies whispered to us by our enemy. God tells us what really is. God is the one we believe.
Lead Me On . . .
And then lead us on. Where do we go? What do we do? There are thousands of tributaries just around the bend. This prayer is simply: God, make us know the way.
We all are called to productivity, meaning that we all will say things and do things and decide things. Praying this second piece of Psalm 143:8 starts the day by demolishing the idea that we have it all figured out. God must give us the wisdom we lack. He must lead us by his sovereign hand. He must because we renounce the ruinous attempt to control our own world. He is our God. This is his world. We lift up our souls to him.
This prayer is hope of the deepest kind, first elating us by the wonder of God’s love at Calvary and then humbling us by the truth that we never stop needing him. There is his definitive, rock-solid demonstration of covenant love and unfailing faithfulness. And then there is his nearness now, his grace today and guidance tomorrow.
Let me hear and lead me on is the best way to start the day.
Let me hear in the morning of your steadfast love,
for in you I trust.
Make me know the way I should go,
for to you I lift up my soul.
Recent posts from Jonathan Parnell:
The Thumbtack of Faith [Desiring God Blog] 2013-10-12 01:00

At bottom, the difference between faith in God and all other alternatives is a choice: to believe or not to believe. But this is no blind leap of faith.
Faith is not irrational.
Faith is not insanity.
Faith is not stepping into the void.
Faith is sanity.
Faith is choosing to see what is actually there.
Faith is the choice to embrace life, the world, God.
Certainly, faith is not exhaustive knowledge or complete understanding. Faith believes certain things that are unseen. But we do not believe the unseen things based on nothing. Faith is not a shot in the dark. Faith is not a good guess.
Faith sees the stars and gapes in wonder. Faith sees a little baby in her mother’s arms and blinks back tears of astonishment. Faith sees even evil, mind-numbing atrocities and aches with revulsion. But these realities do not add up to nothing. They are parts of a story, lines in a poem, and the punch line is God, a good and loving Creator, and a world bracing with beauty, slashed and cracked with evil and sin.
That’s not a stretch. That’s right there in your face, every day.
That means that the choice to not believe, the choice to turn away from God is the suppression of these truths (Romans 1:18). In other words, it is not a rational decision. It is a form of insanity.
But to say that refusing to believe in God is insanity is not the same thing as saying it is not understandable. It is an understandable mistake. It’s understandable because it’s the kind of decision that requires telling the truth. And truth telling has a way of shining light into the corners of peoples’ lives that is highly uncomfortable. This is why our rejection of the light is often multilayered. People build complex psychological and emotional barriers to the truth. They are still culpable. They are still responsible, but it’s understandable because there’s sin down in those corners. It’s dark down there.
To admit that the world has a Creator is to admit that this world has a reason, a purpose. To admit that there is a Designer is to admit that there is a moral order, a functional order, a right and a wrong, a better and worse way to live life. To admit that this world was created is to admit that you have a responsibility for how you have treated others, how you have lived. And the standard for judging your actions is not you. It’s outside of you. And everybody knows instinctively that they have fallen short of the glory they were made for.
So for many, it’s simply unthinkable to believe in God, to believe in a Creator. It’s unthinkable because that would demand thinking certain thoughts that might lead to other conclusions that would eventually imply guilt and responsibility for that guilt. It’s easier to maintain a vague guilt, a vague notion of nobody’s-perfect, and all against the infinite void of evolutionary chaos, which keeps everything sufficiently blurred — we can’t be certain who’s really at fault, so don’t worry about it too much.
As it turns out there’s a pretty sizable army of these relativistic warriors at the moment. The universities are filled with English and Philosophy professors that function as the drill sergeants for this host of guilt-soothers. And the science labs serve up the mysteries, the sacraments of unbelief, insisting that students practice scientific methodologies without actually pressing them into the corners. Pretend the world is ordered. Pretend that logic is meaningful. Pretend that observable phenomena communicate the grace of certainty.
But not too much.
Don’t ask questions about where it all came from. Don’t ask about beauty. Pretend that the little baby in its mother’s womb is just a mass of protoplasm. It could be a tumor. And if you feel that you are a woman trapped inside a man’s body, that’s okay too. No matter that the only scientifically observable phenomenon is the fact that you’re a sexual predator. We will pass laws, and soon you will be able to use whatever locker room you like.
This is insanity, and that’s why faith has the upper hand. Faith is honest about the world. Choosing to believe the latest version of your pagan-approved science textbook, choosing to believe the high priests of atheism, choosing to believe in vague evolutionary relativism is choosing not to see, choosing not to think, choosing to ignore what is right there in front of everyone.
We don’t know which chapter of the story we’re in. We may have another fifty or two hundred years of this kind of cultural insanity. But be assured, we are not playing on equal footing. It’s not like we all squint into the void, and some of us believe in God and His Word and His way of life and some of us squint into the void and say it’s a lot more complex and muddled and who’s to say?
No, we’re not squinting into the void. We’re looking at waterfalls gushing with life. We’re watching the sun sink into a vast ocean, bleeding with beauty. We’re watching the magical glory of a woman, making another person inside of her.
Which means the citadels of unbelief are a facade. We’re not up against a fortress of steel, we’re up against a fortress of balloons. And though they glare down at us through peer-reviewed spectacles, using words like ‘reason’ and ‘logic’ and ‘studies-show,’ we can pull out the thumbtack of faith.
Faith sees the world as it actually is. Faith sees the beauty. Faith sees the glory. Faith sees the art, the story, the goodness, and yes, faith sees the evil in the face of it all and knows that something has gone wrong and we have all become part of the problem.
But when the gospel comes, when Jesus comes, He isn’t talking about some other universe, some kind of alien heaven. He’s talking about this world. He’s talking about this beautiful place, and He’s come to forgive our sins, to heal the brokenness, to raise the dead, to restore the glory. And faith sees that. And that’s how faith overcomes the world (1 John 5:4).
Previous post from Toby J. Sumpter:
How to Humbly Give and Receive Correction [Desiring God Blog] 2013-10-11 01:00

Because we struggle so much with pride, correction can be difficult to give graciously and difficult to receive graciously.
That’s one reason to be very thankful for Exodus 18. God is so kind to have Jethro and Moses give us a clinic on what humble correction looks like on both sides.
At this point in the story, Jethro, Moses’s father-in-law, had escorted Moses’s wife (his daughter) and two boys to rejoin the wild wilderness adventure and heard first hand all the amazing things that God had done for Israel through Moses. Jethro burst into praise and proclaimed God’s supremacy (verses 10–11).
Then Jethro observed his son-in-law at work. Moses was clearly an extraordinary prophet, leader and judge. But there was a problem. Moses spent his whole day judging one dispute after another. Pending cases were backing up. Jethro could feel the mounting frustration and draining fatigue.
Here’s where the clinic begins.
When Moses finally took a break, Jethro asked him a clarifying question: “Why do you sit alone, and all the people stand around you from morning till evening?” (verse 14).
Asking this question was wise and kind. Jethro didn’t jump to a conclusion based on his own perspective. He asked first.
This gave Moses a chance to explain what he was doing and why (verses 15–16): The LORD instructed Moses regarding the law, and Moses’s job was to teach the people and help them apply it to their particular situations. That explanation was helpful.
Understanding this, Jethro said to Moses, “What you are doing is not good. You and the people with you will certainly wear yourselves out, for the thing is too heavy for you. You are not able to do it alone” (verses 17–18).
Jethro was frank: “what you are doing is not good.” No beating around the bush. But Jethro was also gracious. Defective systems can undermine the best mission. His goal was to lift a burden, not tear down intentions.
Notice that Jethro’s critique wasn’t ad hominem. He didn’t say, “Moses, you’re a lousy leader. It shouldn’t take an administrative genius to see that your system doesn’t scale. Do you think you’re qualified to lead two million people?”
No. Jethro’s goal wasn’t to undermine Moses leadership but undergird him. He observed a problem, sought to understand it, identified the core weakness, and offered a helpful solution (verses 19–23). Jethro aimed to increase the effectiveness of Moses’s time use and the meeting of people’s needs.
Now note Moses’s remarkably humble response: “So Moses listened to the voice of his father-in-law and did all that he had said” (Exodus 18:24).
Moses didn’t bristle defensively at Jethro. He didn’t brush him off as an outsider who didn’t understand the organization. He didn’t try to save face by lying that he’d been thinking about doing that very thing himself. And he didn’t pull spiritual rank on Jethro by reminding him who of the two of them heard directly from God more.
No. Moses gratefully received and immediately implemented Jethro’s counsel.
Even though Moses frequently received immediate verbal direction from God, he was not narrow in his understanding of how God speaks and directs. Since God ruled everything he could just as easily direct him through a father-in-law as from a cloud.
If God used Jethro’s correction to direct Moses to greater effectiveness, how much more should we be humbly listening for God’s direction in the correction of those he sends to us?
Jethro’s correction wasn’t just God’s provision for Moses, it was also God’s provision for the needs of thousands of people. When God brings correction to us through the loving observation of someone else, it’s a gift, but not only for us. It’s often for many others as well. If we pridefully resist correction, we are likely plugging up a channel of grace to others. There’s more at stake in our humility than we realize.
So to sum up the lessons from the Exodus 18 Correction Clinic:
When giving correction:
When receiving correction:
Correction is a form of the Lord’s discipline. And Proverbs 12:1 says, “Whoever loves discipline loves knowledge, but he who hates reproof is stupid.” May the Lord help us to love knowledge today.
Recent posts from Jon Bloom:
Meet Grace’s Masterpiece [Desiring God Blog] 2013-10-10 12:59

Do you find theology intimidating? Does it sound distant, cold, academic?
Maybe you’ve known people who are really into theology and don’t seem to love Jesus much, or don’t seem to go to the hard places where poverty, suffering, and unbelief live. So you stopped calling after a lousy first date.
But what if your stereotype of theology really isn’t theology? After all, at its simplest, to do theology is to know more and more about God. That doesn’t sound boring or dangerous at all. Theology is the great social network of knowing God. As God, by his Spirit, applies his words from all over the Bible to our soul, imagination, and worldview, we meet him, not just abstract ideas.
There are dangers of unhealthy distraction or obsession or pride, but there’s nothing more important about us than how we think about God. And not just how we think about his love for us, but what we know about who he is and how he works. After all, how much can we love a God we don’t know very well?
So let’s try an exercise. What has God given us to make us more like himself? You might be able to define sanctification, but how would you describe it to someone? How do you picture sanctification? Better, how would you paint it? Meet Grace.
The painting was so beautiful it haunted her. She couldn’t walk by without stopping.
It hung above their fireplace — two love-bird cardinals on a branch beside a nest in a heavily wooded area sometime late October or early November. A few red and yellow leaves remained, but only a few. If you looked close enough, you’d see the old, gray shed almost hidden in the upper right-hand corner.
Her dad had painted the piece several years ago — in fact, he was always painting. He’d see something on a drive or while walking in their neighborhood or flipping through a magazine, and he’d bring it to life right in front of Grace’s ten-year-old light brown eyes with just a brush and a handful of colors.
She wanted to be like him, to create like he created — to be conformed to her favorite artist, from one degree of beauty and creativity to another. Her parents would let her play with her own paint, and she would try to imitate the fireplace birds or the flowers in her bedroom or the lighthouse in the bathroom. But nothing she made ever satisfied her like his brush could.
One day, having tried and failed, and tried and failed, she asked Daddy for help. Like most dads, he couldn’t wait to say yes. He set up an easel in his study, placed her chair right in front, his behind hers, and gave her a small palette of colors — just green, red, and yellow with a little white and black.
So she began painting. While she was holding the brush, it was hardly her doing the painting. Sure, he never touched the canvas without her, but his mark was in every stroke. He pointed and motioned and taught and even moved her hand with his own. First a tree — trunk, branches, leaves, even a bluebird — then another and another and a trail between them, like the ones they had hiked together last summer.
She saw the scene being born and loved it. It wasn’t easy, and it wasn’t anything as good as her dad’s, but it was like his, and it was hers, and they had done it together.
That night she sat staring at her art, treasuring the trees, but seeing more and more flaws. It needed something more — details in the branches, more colors, more life. Tears welled up in her young eyes as she realized just how much was missing. As she studied the painting, longing for more, she fell asleep.
When Grace woke up the next morning, which seemed like no time at all, she found something very different in front of her. This painting reminded her of hers, but it had been changed, strangely made new. She saw all her own strokes, the hours she had put in yesterday, but there was so much more now.
Where there had been two or three shades of green before, now there were two or three hundred, all working together to breathe fullness into her trees. The birds, once flat, were brought to life — made to look real and soft and even hungry. You could almost hear them through the canvas.
Everything she had hoped to see and much more filled her heart far beyond all her imagination had made the night before. Her eyes were wide and full. She carefully searched every inch, looking for any flaw or lack in this little forest. She knew the beauty was hers, and yet it was so much more than hers.
She looked and looked at the painting with wonder. And as she did, her dad walked by holding his morning coffee, smiling as he did. And as she saw him, the same man who had sat with her the day before, she loved him more than ever, because only one was able to do what had been done that night. Only one could make her work this beautiful, this perfect.
It was her brush. His masterpiece. Our sanctification.
It is God who works in you, both to will and to work for his good pleasure. –Philippians 2:13
We all, with unveiled face, beholding the glory of the Lord, are being transformed into the same image from one degree of glory to another. –2 Corinthians 3:18
When he appears we shall be like him, because we shall see him as he is. –1 John 3:2–3
Love God with Your Everything [Desiring God Blog] 2013-10-10 01:00

Love. There are few things so universal and yet so challenging. Love for God. “The most important commandment,” says Jesus (Mark 12:29–30), and one that both the old and new covenants portray as necessary to enjoy God’s sustained favor. As Moses asserted, Yahweh “keeps covenant and steadfast love with those who love him and keep his commandments, to a thousand generations,” but he “repays to their face those who hate him, by destroying them” (Deuteronomy 7:9–10). Similarly, Paul declared that “all things work together for good” only for “those who love God . . . who are called according to his purpose” (Romans 8:28).
Some have tagged the Supreme Command of Deuteronomy 6:5 the “all-command,” because of the three-fold “all” — “You shall love the Lᴏʀᴅ your God with all your heart and with all your soul and with all your might” (ESV). There is no room here for divided affections or allegiance. As Jesus said, “No one can serve two masters” (Matthew 6:24). If indeed there is one God who stands supremely powerful and valuable (Deuteronomy 6:4), this demands a supreme and total loyalty from you and me, a loyalty that starts with the heart.
While surprising to some, the old covenant recognized that a spiritual relationship with God begins from within, with a proper disposition toward the preeminent savior, sovereign, and satisfier. From the heart “flow the springs of life” (Proverbs 4:23), and without one’s will, desires, passions, affections, perceptions, and thoughts rightly aligned, the life of love is impossible. Therefore Moses calls Israel to “know . . . in your heart” that God disciplines like a father his son. He urges God’s people to “lay it to heart” that there is no God besides Yahweh (Deuteronomy 4:39–40) and to ensure that his words “be on your heart” (Deuteronomy 6:6), thus anticipating the miraculous heart-work that the new covenant would realize (Jeremiah 31:33).
Along with our hearts, we are called to love Yahweh with all our soul. In the first five books of the Old Testament the “soul” refers to one’s whole being as a living person, which includes one’s “heart” but is so much more. For example, in Genesis 2:7 we are told that “Yahweh God formed the man of dust from the ground and breathed into his nostrils the breath of life, and the man became a living soul creature” (cf. 9:5). Elsewhere corpses are called “dead souls,” which simply means the person, once alive, is now dead (Leviticus 21:11), and Yahweh promises that his “soul [i.e., his being] shall not abhor” all who follow his lead (Leviticus 26:11). In light of these texts, it seems Moses starts with a call to love God from within and then moves one step larger saying that everything about us as a person is to declare Yahweh as Lord. So we are to love God with our passions, hungers, perceptions, and thoughts. But we are also to love him with how we talk, and what we do with our hands, and how we utilize our talents, and how we react to challenges –– our entire being is to display that we love God.
What then is the meaning of loving God with our “might”? The word translated “might/strength” in Deuteronomy 6:5 usually functions as the adverb “very” in the Old Testament (298x). The noun version occurs in Deuteronomy and in only one other place, which itself is just an echo of our passage. In 2 Kings 23:25 we are told that King Josiah “turned to Yahweh with all his heart and with all his soul and with all his might.” So if the word usually means “very,” what would it mean to love the Lᴏʀᴅ will all our “very-ness”? Interestingly, the Greek translation of this word is “power.” The Aramaic translation is “wealth.” Both of these may actually be pointing in the same direction, for the strength of a person is not simply who he is but what he has at his disposal.
Think with me: If Moses’s call to love Yahweh starts with our heart and then moves out to our being, could not our “very-ness” be one step bigger and include all our resources (see Block, Deuteronomy, 183–84)?
This means that the call to love God is not only with our physical muscle but with everything we have available for honoring God — which includes our spouse, our children, our house or dorm room, our pets and wardrobe and tools and cell phones and movies and music and computers and time.
So are we on target reading it this way? The context of Deuteronomy 6 would suggest we are. Verses 6–9 stresses that treasuring God’s oneness and uniqueness needs to be personally applied to our lives (verses 6, 8). It needs to impact relationships (verse 7), and what goes on at home and in the work place (verse 9). This means that the covenant love we’re called to must be wholehearted, life-encompassing, community impacting, exclusive commitment to our God. And this God is our God only because he has now revealed himself to us in the person of his Son. This kind of love we should have for him doesn’t exist apart from love for Jesus — for Jesus and the Father are one (John 10:30).
This truth means that every closet of our lives needs to be opened for cleaning, and every relationship in our lives must be influenced. This call to love God this way destroys any option of being one person at church and another person on a date. What you do on the internet needs to be just as pure as what you do in Bible-reading. The way we talk to our parents needs to be as wholesome as the way we talk to our pastors. There needs to be an authentic love for God that starts with God-oriented affections, desires, and thoughts, that permeates our speaking and behavior, and then influences the way we spend our money and how we dress, and drive, and our forms of entertainment.
Whether we’re eating or singing, jogging or blogging, texting or drawing, love for Yahweh — the one true triune God — is to be in action and seen.
Pornography: The New Narcotic [Desiring God Blog] 2013-10-09 16:51

The new narcotic. Morgan Bennett just published an article by this title. The thesis:
Neurological research has revealed that the effect of internet pornography on the human brain is just as potent — if not more so — than addictive chemical substances such as cocaine or heroin.
To make matters worse, there are 1.9 million cocaine users, and 2 million heroin users, in the United States compared to 40 million regular users of online pornography.
Here’s why the addictive power of pornography can be worse:
Cocaine is considered a stimulant that increases dopamine levels in the brain. Dopamine is the primary neurotransmitter that most addictive substances release, as it causes a “high” and a subsequent craving for a repetition of the high, rather than a subsequent feeling of satisfaction by way of endorphins.
Heroin, on the other hand, is an opiate, which has a relaxing effect. Both drugs trigger chemical tolerance, which requires higher quantities of the drug to be used each time to achieve the same intensity of effect.
Pornography, by both arousing (the “high” effect via dopamine) and causing an orgasm (the “release” effect via opiates), is a type of polydrug that triggers both types of addictive brain chemicals in one punch, enhancing its addictive propensity.
But, Bennett says, “internet pornography does more than just spike the level of dopamine in the brain for a pleasure sensation. It literally changes the physical matter within the brain so that new neurological pathways require pornographic material in order to trigger the desired reward sensation.”
Think of the brain as a forest where trails are worn down by hikers who walk along the same path over and over again, day after day. The exposure to pornographic images creates similar neural pathways that, over time, become more and more “well-paved” as they are repeatedly traveled with each exposure to pornography. Those neurological pathways eventually become the trail in the brain’s forest by which sexual interactions are routed. Thus, a pornography user has “unknowingly created a neurological circuit” that makes his or her default perspective toward sexual matters ruled by the norms and expectations of pornography.
Not only do these addictive pathways cause us to filter all sexual stimulation through the pornographic filter; they awaken craving for “more novel pornographic content like more taboo sexual acts, child pornography, or sadomasochistic pornography.”
And it gets worse:
Another aspect of pornography addiction that surpasses the addictive and harmful characteristics of chemical substance abuse is its permanence. While substances can be metabolized out of the body, pornographic images cannot be metabolized out of the brain because pornographic images are stored in the brain’s memory.
“In sum,” Bennett writes, “brain research confirms the critical fact that pornography is a drug delivery system that has a distinct and powerful effect upon the human brain and nervous system.”
None of this takes God by surprise. He designed the interplay between the brain and the soul. Discoveries of physical dimensions to spiritual reality do not nullify spiritual reality.
When Jesus said, “I say to you that everyone who looks at a woman with lustful intent has already committed adultery with her in his heart” (Matthew 5:28), he saw with crystal clarity — the way a designer sees his invention — that the physical eye had profound effects on the spiritual “heart.”
And when the Old Testament wise man said in Proverbs 23:7, literally, “As he thinks in his soul, so is he,” he saw with similar clarity that soul acts create being. Thinking in the soul corresponds to “is.” And this “is” includes the body.
In other words, it goes both ways. Physical reality affects the heart. And the heart affects physical reality (the brain). Therefore, this horrific news from brain research about the enslaving power of pornography is not the last word. God has the last word. The Holy Spirit has the greatest power. We are not mere victims of our eyes and our brains. I know this both from Scripture and from experience. And I will write more about it next Tuesday.
Related resources:
Sexual Sin in the Ministry by Harry Schaumburg
Fake Love, Fake War: Why So Many Men Are Addicted to Internet Porn and Video Games by Russell Moore
Porn, Pride, and Praise: An Interview with Heath Lambert, Authors on the Line
All Lewis Conference Media Now Available [Desiring God Blog] 2013-10-09 11:39

Whether you’ve read the Chronicles of Narnia, the Space Trilogy, Mere Christianity, or other works by C.S. Lewis, you’ve probably wanted to ask him some questions. While we can’t give you the man himself — he died 50 years ago this fall — we might have your answer. Or 23 of them.
Our recent national conference dove deeply into the life, heart, and influence of Lewis, celebrating all we’ve learned through him and asking the hard questions of his writing. It’s a collection of talks that offers a big, thorough look at Lewis — his God, imagination, books, theology, friendships, worship, and more.
Please enjoy and share all the audio and video below free of charge.
John Piper: “C.S. Lewis, Romantic Rationalist: How His Paths to Christ Shaped His Life and Ministry”
Philip Ryken: “Inerrancy and the Patron Saint of Evangelicalism: C.S. Lewis on Holy Scripture”
Douglas Wilson: “Undragoned: C.S. Lewis on the Gift of Salvation”
Kevin Vanhoozer: “In Bright Shadow: C.S. Lewis on the Imagination for Theology and Discipleship”
Speaker Panel: “Uncovering Lewis’s Uncommon Influence”
Randy Alcorn: “C.S. Lewis on Heaven and the New Earth: God’s Eternal Remedy to the Problem of Evil and Suffering”
John Piper: “What God Made Is Good — And Must Be Sanctified: C.S. Lewis and St. Paul on the Use of Creation”
N.D. Wilson: “Myth Wars: C.S. Lewis vs. Scientism”
Colin Duriez: “The Friendship of C.S. Lewis and J.R.R. Tolkien”
Lyle Dorsett: “C.S. Lewis and the Care of Souls”
Joe Rigney: “Live Like a Narnian: Christian Discipleship in C.S. Lewis’s Chronicles”
Sam Storms: “How Lewis Changed the Way I Worship”
Noel Piper: “Latecomer to Narnia”
N.D. Wilson: “The Lie of Realism”
Joe Rigney: “Lewis Against Hipsters — And Those Who Despise Them”
Jonathan Parnell: “Pizza! Pizza! Lewis at Little Caesars”
David Mathis: “Fed up with Life and Ready to Write”
Tony Reinke: “Jack’s Typewriter”
Matt Reagan: “A Child’s Heart and a Grown-up’s Head”
Lyle Dorsett: “Longing in Lewis’s Life and Writing”
Colin Duriez: “Was Lewis a Revolutionary or Dinosaur?”
Devin Brown: “A Quick Look at the Best of ‘Screwtape’”
Dan Dewitt: “Why Lewis Wouldn’t Write for ‘Christianity Today’”
Download the song list from the sessions of corporate worship (PDF).
15 Quotes from the C.S. Lewis Conference [Desiring God Blog] 2013-10-09 01:20

As we finish uploading all the audio and video of the C.S. Lewis conference online for you in the next few hours, here’s a sampling of fifteen quotes from the conference talks that caught our attention. These will give you a flavor of what to expect when you enjoy the conference recordings for yourself (soon).
UPDATE: All the conference media is now available.
Douglas Gresham, conference introduction video —
This year the conference is titled, “The Romantic Rationalist: God, Life, and Imagination in the Work of C.S. Lewis.” Jack, my stepfather, would be pleased by your organization’s name: Desiring God. For perhaps the most important element that led to his own conversion was a strange and powerful longing that he felt throughout his life. Though for many years he did not know what this was a longing for, in the end, he came to see that all along it had been a desire for God.
John Piper, first plenary, “C.S. Lewis, Romantic Rationalist: How His Paths to Christ Shaped His Life and Ministry” —
What was Lewis doing in all his works? He was pointing. He was unveiling. He was depicting the glory of God in the face of Jesus. He was leading people to Christ. The two paths he knew best were the paths of romanticism and rationalism — longing and logic. So these are the paths on which he guided people to Christ.
Joe Rigney, seminar, “Live Like a Narnian: Christian Discipleship in C.S. Lewis’s Chronicles” —
We are, all of us, en-storied creatures, living our lives in a narrative, which means our lives have directions, trends, and trajectories. And Lewis is mindful of the fact that these trajectories are governed by an Author who is not mocked, who tells us that we will reap what we’ve sown. … Lewis is clear: we are always sowing the seeds of our future selves. We are embarked, heading in a particular direction, and sooner or later we are bound to end up there. Edmund reminds us that we might not like the destination at the end of our road. When it comes time to reap, we may find ourselves tied to a tree with a dagger at our necks. But, of course, Edmund’s story isn’t a tragedy. Yes, it’s true; reaping always follows sowing, like night follows day. But in this case, Aslan reaps what Edmund has sown. Edmund’s treachery, Edmund’s spite, Edmund’s beastliness is thrown onto Aslan and the Lion bears it away in his death at the Stone Table. This is the Magic of substitution, the Deeper Magic that turns traitors into kings, that turns beastly boys into just and wise men, the kind of magic that changes our stories forever.
Devin Brown, small talk, “A Quick Look at the Best of ‘Screwtape’” —
Long before there was Narnia there was Screwtape. For the one and only time in his life, C.S. Lewis appeared on the cover of Time Magazine (1947), pictured with a devil over his shoulder. The Screwtape Letters is the book that put him on the cover.
Randy Alcorn, plenary, “C.S. Lewis on Heaven and the New Earth: God’s Eternal Remedy to the Problem of Evil and Suffering” —
Why does Reepicheep the mouse want to go to Aslan’s country? To be with Aslan. It is all about Jesus. We are made for a person and a place. Jesus is the person, heaven is the place — but the place is meaningless if Jesus isn’t there.
C.S. Lewis on Tolkien’s The Lord of the Rings, as quoted by Piper in his first plenary —
I’ve never met Orcs or Ents or Elves — but the feel of it, the sense of a huge past, of lowering danger, of heroic tasks achieved by the most apparently unheroic people, of distance, vastness, strangeness, homeliness (all blended together) is so exactly what living feels like to me. Particularly the heart-breaking quality in the most beautiful places, like Lothlorien.
Kevin Vanhoozer, plenary, “In Bright Shadow: C.S. Lewis on the Imagination for Theology and Discipleship” —
For Lewis, waking is a way of describing conversion — a coming to new life. The Christian life is all about wakefulness. Theology describes what we see when we are awake, and discipleship is about staying awake. The sad truth is many of us are at best only half awake. We think we’re engaged with the real world — the world of stock markets, stock car racing, stockpiles of weapons — but in fact, we’re living in what Lewis calls the Shadowlands. We’re really daydreaming and sleepwalking our way through life, asleep at the wheel of existence. …
Theology ministers understanding, so that we can live out our knowledge of God. Theology is practical, it is all about waking up to the real, to what is, specifically to what is ‘in Christ.’ … The imagination helps disciples act out what is ‘in Christ.’ Theology exchanges the false pictures that hold us captive with truth and disciplines our imagination with sound doctrine.
Phillip Ryken, plenary, “Inerrancy and the Patron Saint of Evangelicalism: C.S. Lewis on Holy Scripture” —
Lewis’s doctrine of Scripture [inspiration/inerrancy] has often been regarded as sub-orthodox. But whatever deficiencies we find, they do not seem to have a devastating effect on his theology as a whole. Typically, such theologians downgrade other doctrines, they back away from the hard sayings of Jesus, or become skeptical about biblical miracles, or dismiss the deity of Christ. Lewis did none of that. He continued to give a robust defense of biblical Christianity.
Plenary panel discussion —
Phillip Ryken: [Contrasted to living authors who deny inerrancy] Lewis is very clear he wants to be in submission to the authority of Scripture. There are some people in the church today; you sometimes get the sense they’re standing a little bit in authority over Scripture. You don’t get that sense with Lewis.
Randy Alcorn: And a lot of people today who are Christian leaders are drifting; they have grown up holding to truths they’re now departing from, their trajectory is away from the gospel. Lewis came from atheism, moving to agnosticism, theism, came to a life-changing faith in Christ — he was growing in his life. He came from a world where he didn’t have the doctrinal reference points. His trajectory was, in my opinion, toward the gospel.
N.D. Wilson, small talk, “The Lie of Realism” —
Lewis wrote fantasy stories because he thought, correctly, that that’s what the world is really like. The lie of realism is that somehow we’ve let people name ‘important fiction’ in which there is no soul, no spirit, no supernatural — “realistic.” In realistic fiction, there can be no magic, no supernatural, no God, no soul to the character. … We’re on a rock, mostly molten lava, flying through outer space at about mach-86 right now, like a yo-yo being swung around a ball of fire in the sky. That’s our setting. What kind of story are we telling? We’re in the sci-fi fantasy section of the bookstore.
Douglas Wilson, plenary, “Undragoned: C.S. Lewis on the Gift of Salvation” —
I don’t feel safe around anything when Jesus is not the Lord of it. Calvinism without Jesus is deadly; it’s fatalism, it’s simply Islam. We need Jesus. When the precious doctrines [of Calvinism] are used to perpetuate gloom, severity, introspection, accusations, morbidity, slander, gnat-stringing, and more, the soul is not safe.
C.S. Lewis as quoted by Sam Storms in his small talk —
Fully to enjoy is to glorify. In commanding us to glorify Him, God is inviting us to enjoy Him.
John Piper, final plenary, “What God Made Is Good — And Must Be Sanctified: C.S. Lewis and St. Paul on the Use of Creation” —
Eating food becomes worship by acts that terminate on God not merely on food. Thanking is for food, but to God. Believing is believing in God and his Son Jesus Christ. Knowing terminates on truth and ultimately on God. Eating is not worship. Eating becomes worship — through knowing and believing and thanking. The created world is not an end in itself. It finds its meaning when people, created in God’s image, use it with a mind that knows God, and a heart that believes in and thanks God. …
I’m suggesting, along with Lewis, that of all the possible ways that God could have revealed the fullness and diversity of the supreme value of his being, he concluded that a physical world would be the best. The material creation was not God’s way of saying to humankind: “I am not enough for you.” It was his way of saying: “Here is the best garden where more of what I am can be revealed to finite creatures. The juiciness of a peach and the sweetness of honey are a communication of myself.”
A Prayer for the Worried Mom’s Heart [Desiring God Blog] 2013-10-08 11:24

Do you ever worry?
I think we can all admit that we do. In fact, we probably worry more than we realize. As a mother, I find myself worrying about my children, about their health, their learning, and whether I can just make it to bedtime each day.
I also find myself worried about paying bills, about my husband’s travel for work, and about that message from my doctor needing to discuss test results with me. My to-do lists keep me awake at night because I fear I’ll forget to do something important. Questions like “what if?” and “should I have?” swirl around my mind, holding me hostage and keeping me chained to my worries and fears.
Worry is a kind of “acceptable sin.” By that I mean worry is one of those sins that everyone does so we don’t often address it. Like gossip, worry is something we all know we aren’t supposed to do, but we often gloss over it and call it something else — something like stress. Especially for women, worry can be expected and in some situations to not worry would seem strange.
But deep down, we want to be freed from the chronic feeling of doom and the expectation of something bad lurking just around the corner. We know that the Bible tells us not to worry, but “what if?” thoughts seem like such a part of us that we don’t know how to stop.
What can we do?
Like oil and water, trust and worry do not mix. To expel worry from our heart, we need to grow deeper roots of trust in God. Time and again in the Psalms, when the writer’s heart was heavy, he turned to look back at all that God had done for him. As the psalmist looked back at God’s faithfulness and his sovereign care for him, he was able to trust God even in the midst of troubling circumstances.
When we look back in our own lives at God’s faithfulness to us, it gives us confidence and hope in his future faithfulness. We look back to our own story of salvation. We see that Jesus died on the cross for our sins, that this is the demonstration of God’s love for us. When worries threaten to seize our heart, we need to remember and dwell on the truth of the gospel. Remembering the cross propels us in faith for what lies ahead.
And as we remember, we need to turn to God in prayer. Hebrews says that because of Jesus, we can come to the throne of grace with confidence, to receive the help we need (Hebrews 4:16). Paul was referring to chronic worry when he wrote in Philippians 4:6, “Do not be anxious about anything, but in every situation, by prayer and petition, with thanksgiving, present your requests to God.” We are to give our worries to God in prayer, trusting him with all our burdens and cares. As a result, we will receive in return the peace we long for, “And the peace of God, which surpasses all comprehension, will guard your hearts and your minds in Christ Jesus” (Philippians 4:7).
You might even pray something like this. . . .
My Papa in Heaven,
I come to you with a heart heavy and full of so many worries and cares. I want to just curl up on your lap and find some peace from the chaos in my life. My worries fill my mind night and day. My stomach is in knots and I can hardly breathe. I feel like I am drained dry; the joy has been sucked right out of me.
But you said to come to you with all my burdens. You said that you will carry them. You tell us you are a rock, a shield, a fortress. I need a rock right now. I need a fortress to run into right now. I need you.
There are so many decisions to make. What if I make the wrong one? So many bad things loom on the horizon, what if I’m not prepared? Help me to focus my heart on you and not on the giants around me. I know that all these worries are keeping me from trusting you. Like Peter, instead of looking toward your face, I am looking around at the waves encircling me.
Forgive me for doubting and not living a life of trust. I believe, but please help my unbelief! I know that when I worry, I am believing a lie that says that I can control what happens in my life. Forgive me for trying to control something I never really had control of. Help me to trust in your word and not the lies.
You sent your Son to carry my greatest burden at the cross. I know that you can handle all that troubles me today. There is nothing too great for you, the earth is your footstool and the wind and rain come and go at your command. Free me of this worry today. Help me to trust the same grace that saved me at the cross to save me from all that weighs me down.
I know that you have a perfect plan for my life. Help me to walk by faith and not by sight. I want to trust in your plan and your love for me. I want to face the unknown future confident that you have it under control. Grant me the grace I need.
Thank you for Jesus and that because of him I can come to you in confidence. You accept me as I am, worries and all. I give them all to you now, in Jesus’s name, Amen.
Related posts from Grace at Home:
How to Be Interesting and Unhelpful [Desiring God Blog] 2013-10-08 01:00

The point of this little exhortation is that, in handling the Scriptures, sanctification and speculation rise and fall in inverse proportion. As speculation increases, sanctification decreases. The more guessing the less blessing.
Few people would give their life for a speculation. Few will gouge out an eye or cut off a hand, because of a guess. Suppositions make weak expositions.
Here’s the sort of thing I have in mind.
Preachers, teachers, and Bible study leaders are sometimes tempted to speculate because the “possibilities” are so interesting.
For example, what about possible appearances of Christ in the Old Testament? When it says God was walking in the Garden of Eden (Genesis 3:8) could that have been Christ? Was Melchizedek really Christ himself in Genesis 14? When “the Lord” appeared to Abraham in Genesis 18 was that a pre-incarnate appearance of Christ? When Jacob wrestled all night with “a man” (Genesis 32:24) was it Christ? Was the fourth person in the fiery furnace (Daniel 3:25) Christ?
Could the splitting of the waters of the Red Sea be explainable by God’s using some cosmic catastrophe to create atmospheric conditions that caused the waters to divide?
Was there a written document containing all the material common to Matthew and Luke but missing from Mark, which we might call Q for German Quelle, meaning source?
Was the later poverty of the church in Jerusalem (Romans 15:26) owing to the misapplication of the early policy of having “all things in common” (Acts 2:44, 4:32)?
Was Paul a widower? Or did his wife leave him when he became a Christian? Did he have children?
Did Mary Magdalene have a crush on Jesus? Did Jesus have to deal with more temptations than we usually think?
When Paul prohibited women from teaching and having authority over men in 1 Timothy 2:12 was there a problem in that church such as women taking authority and teaching, who were unprepared to do so? Was that the only reason Paul prohibited them — they weren’t ready?
My point is that people need solid food, not possible food. They need a sure word from God, not a guess from man. They need a biblical “Thus says the Lord,” not a “Maybe God said.”
A fascinating five-minute homiletic detour into what might have been going in Corinth behind this or that text is a waste of precious time. And I think it trains our people to expect interludes of historical entertainment, and to mistake it for deep insight and spiritual food.
What is really there in the text of Scripture is bottomless, and staggeringly interesting, and provocative. Speculation is not necessary to hold people’s attention. If a pastor finds what might have been more interesting than what is really there in the text, he needs better powers of observation, not better powers of speculation. He needs a better feel for the wonder of what is, than a greater fancy for what might have been.
Two qualifications:
Poetry and preaching are not the same. Illuminating fiction and authoritative exposition are not the same. I love poetry and fiction. These are by nature inventive. They too have their place and their power. But the sanctifying power they have is owing decisively to the deeper truths they convey, not the imaginative structures that convey them.
When a text of Scripture is apparently contradictory, and there is little agreement on what the solution is, it is helpful for people to see one or two possible and plausible solutions. These will be more or less speculative. We tell our people we are not sure of the answer. We don’t want them to take our guesses as God’s word. But we offer our guesses so that they can see at least the possibility that there is a solution here rather than a contradiction.
So I say again, the crying need in the pulpit and the classroom is not to spend time speculating about what might have been the case, but to dig deeper into what is really there in the text. Most of us are still scratching the surface.
If we know for sure something that’s not in the Bible, and we see that it sheds true light on the Bible, that’s another matter. Let it be so. But my sense is that the secondary literature is no easier to interpret than the Bible. Which means that the secondary texts are no more clear than the biblical texts they supposedly illumine. It is a harmful thing to teach seminarians to see the Bible as needing help from outside, while failing to see that the outside documents also need help from the outside.
Pastors and teachers have very limited time for study. The ocean of contextually understandable Scripture is bottomless. My plea is simple: dive deeper in what’s there.
See translated versions:
Recent posts from John Piper:
The Biggest Spectacle at Catalyst [Desiring God Blog] 2013-10-07 12:13

ATLANTA — It was our first time at Catalyst, and we were prepared for some highly produced craziness.
They have fired a man from a cannon, belly-flopped another guy 30 feet into a kiddie pool, offered camel rides onsite, and set ridiculous world record after ridiculous world record — including bubbles, Frisbees, and whoopee cushions. Not exactly where you expect to find our 67-year-old Pastor John.
But the biggest spectacle at Catalyst, or any other place in the world, is Jesus Christ, the sinless Son of God crucified for sinners. And John Piper had the amazing opportunity on Thursday to speak of this Jesus to more than 12,000 young, gifted leaders gathered at the Gwinnett Arena in Atlanta.
In summary, his message makes clear that you were created to display God. You are made in the image of Another, and all of us have failed to fulfill our one great purpose. But Jesus — the great spectacle of history — has purchased our re-creation with his death at Calvary. And now the best news in the whole world is that, in Christ, God’s unfailing pursuit of his glory has become his unfailing pursuit to make you happy in himself forever.
As many might imagine, it was a sudden shift in tone from the leadership-oriented talks characteristic of the Catalyst movement, but it became clear that this message provides the ground and substance of leadership and ministry — a substance that can never be replaced by savvy techniques, entertaining stunts, or skinny jeans.
Stream or download the 42-minute message, “Identity and Desire.”
Recent new messages from John Piper:
It’s Just Complicated — And Very Simple [Desiring God Blog] 2013-10-07 01:00

The Christian life can be so complex — and oh so very simple.
That we would use such a fancy word as sanctification betrays the complexity. But that defining such a big word could be so easy hints at the simplicity.
The word sanctification is built on the Latin sanctus, meaning “holy.” Sanctification is the theological term we Christians often use for the process of being made holy. For the Christian, whose standard of perfect human holiness is Jesus, the God-man, sanctification is essentially becoming more like Jesus — being “conformed to the image of his Son,” as Romans 8:29 puts it.
Christian growth, or maturation, is another way to define sanctification. It’s a big word for the little-by-little progress of the everyday Christian life. That much is simple. But what it encompasses is enormous: how every professing Christian should live, where our holiness is heading, how fast the progress should be, and how it happens in real life. Here’s where it gets complicated and controversial.
Sanctification talk gets prickly quickly because it immediately involves so many massive realities in the Christian worldview and their coming together in daily life: grace and works, law and gospel, faith and the Holy Spirit, Christian obedience and pleasing God, love and good deeds, and much more. And sanctification gets very personal — it’s about the details of your life.
The stakes are high. Weak spots in our theology will turn up, before long, in our understanding, and pursuit, of sanctification. It doesn’t take much time before a wacky doctrine elsewhere begins to mess with our doctrine and practice of holiness. True Christian theology is a seamless garment, and every doctrine eventually relates to every other, but sanctification seems to call the question faster than the others, and has the tendency to accentuate our problem areas.
Because of the inherent complexity of sanctification, involving all these moving pieces, there is a great temptation to oversimplify things. Because sanctification with all its tentacles feels like an octopus larger than we can comfortably tame, we tend to prefer our own little theological house pets that we can easily train and remain captain of. It’s nice to have a slogan that can keep it simple for stupid humans and make us feel like we’re in control.
Enticing as it sounds — and convicting as it may be to hear about if you’ve tried it — the well of sanctification reductionisms soon runs dry. “Let go and let God” — it won’t be long before that creates some problems. “Simply obey” — that won’t do it either. “Just get used to your justification” — attractive, yes, but there’s another reductionism at work here. Even “union with Christ as the key,” close as it may be, falls short of capturing the full picture.
It’s as if we find the biblical data to be just too numerous and complicated, and what we really need is to search for sanctification’s holy grail. It must be out there somewhere — surely, there’s some quick fix, some theological secret to discover, some doctrinal key that unlocks what holiness really is and how to have it.
But if there’s any key to sanctification, it’s this: abandon your search for the key. At least abandon the search for a shortcut. Let your quest for the holy grail of sanctification end right here and right now, and commit to a sanctification not of only, but of all — all the Scriptures, all of Christian theology, all the Bible’s salvific pictures, and, ultimately, all of Jesus.
What is needed for Christian sanctification is not some silver-bullet doctrine or fresh slogan or new overriding emphasis, but the whole of the Bible, theology, and Jesus. The same Jesus who is our righteousness for justification is the same Jesus who is our holiness for sanctification — and is the same Jesus we’re united to by faith to receive all God’s priceless graces.
By virtue of our Spirit-powered, by-faith union with Jesus, we have the new-creation spiritual life of regeneration, and the righteousness of justification, and the holiness of sanctification, and the familial affection and privilege of adoption, and the honor of glorification. This is big. It gets complicated. There are so many ducks that it’s hard to get them all in a row — and that’s just the way God would have it. After all, he is the sanctifier, not we. He would rather we always lean on him for holiness than supposing we have it figured out.
But even in all the complexity, there is a point of focus that can help us get our bearings and give us some semblance of simplicity. The beginning and end of Christian sanctification is none other than Christ himself. There is an initial relationship with Jesus that first sets us apart as definitively sanctified, and gets the gears going for our ongoing sanctification, but a deepening relationship with Jesus is the heart of sanctification, and knowing Jesus is the great goal of our sanctification.
Jesus is not only the preeminently sanctified one and the one who empowers our sanctification by his Spirit, but also he is the one whom the whole of our sanctification is shaping us to know forever (Ephesians 5:26–27). Knowing Jesus drives us onward in sanctification now (Philippians 3:8), and knowing Jesus is the eternal life that sanctification fits us for (John 17:3).
The greatest blessing of salvation is not mere forgiveness. It’s not just justification and being declared righteous. It’s not new birth. It’s not even sanctification. It’s not just the privilege of being united to him, but being united to him serves the greater goal of enjoying him.
The greatest blessing of redemption is Jesus himself. All aspects of the Spirit’s subjective application to us, and all of Jesus’s objective accomplishments for us, conspire to this one great end: knowing Jesus, enjoying Jesus, admiring Jesus, and treasuring Jesus for all eternity.
David Mathis writes on “The Search for Sanctification’s Holy Grail” in Desiring God’s newly available Acting the Miracle: God’s Work and Ours in the Mystery of Sanctification, which includes contributions from John Piper, Kevin DeYoung, Ed Welch, Jarvis Williams, and Russell Moore. The book is now available in softcover, as well as a free PDF, from Crossway Books and Desiring God.
Does Success Start on Sunday? [Desiring God Blog] 2013-10-06 01:00

In the outskirts of the city, on a road that’s walked as much as driven, a typical car brakes at a red light.
It is the kind of car so typical that the actual model stays blurry in memory. It is the kind of braking so natural that the driver must know this block. Everything in the scene fits: the worn road, the red light, the common car, but not the bumper sticker. That is a different story, with its weathered corners and sunbaked background accenting a phrase in all-caps Comic Sans: “Success Starts on Sunday.”
There is also a church name, one as typical as the car, listed below the slogan in smaller letters. And now we get it. Said straight, the shiny message on this tattered sticker goes:
If you want success then go to church.
We’ve seen this before. Whether the devil’s out to get us or that big promotion needs reeling in, the most necessary thing for us to do, as this initiative would say, is to be at church today. Prophesying peril is one way; promising prosperity is another — and it’s actually more insidious.
A lot more folks like the sound of success over the sensationalism of Satan-talk. Whole movements are built on that, in fact. And even if we’re not driving to a basketball arena this morning, there’s still a chance we’ll be gigged by that gimmick. Does success really start on Sunday? Does it?
The real question is, “What do we want?”
If what we’re really after is to drive a nice vehicle or wear tailored suits or pad the 401k or grow business clientele or raise healthy kids or feel good about ourselves, then church attendance is not our problem. Idolatry is.
And if idolatry is our problem, we don’t need success, we need dust and ashes. We need to be made alive. Sunday, indeed, brings us hope for that, but it’s not this Sunday. It was a Sunday two thousand years ago when the earth shook and a large stone was rolled away. Jesus Christ, our Savior, crucified for our sins, dead in our place, was raised in power to overcome every obstacle to our everlasting joy in God.
Truth is, success doesn’t start on Sunday. Victory was accomplished on Sunday, and therefore we worship God alone, today and every other day of the week.
Recent posts from Jonathan Parnell:
Smiting the Green-Eyed Monster [Desiring God Blog] 2013-10-05 01:00

It’s everywhere you turn, yet so easily keeps itself hidden.
It’s pervasive in advertising and steers the course of political debates. It lives in all our hearts, but seems so petty that we’re unwilling to admit its presence.
But in God’s economy, envy is sinful and dangerous. It makes frequent cameos in the New Testament lists of nastiness (Mark 7:22; Romans 1:29; 1 Timothy 6:4; Titus 3:3). We hear that “love does not envy” (1 Corinthians 13:4), that sadly some “preach Christ from envy” (Philippians 1:15), and, worst of all, that “it was out of envy” that they delivered up Jesus to be crucified (Matthew 27:18; Mark 15:10). And so we’re directed to fight it (Galatians 5:26; 1 Peter 2:1).
Envy, says Joe Rigney, is a feeling of unhappiness at the blessing of others. It’s different than jealousy, which orients on what we ourselves own and is not always a sin. Envy orients on others and their blessing. And it hunts in a pack, keeping company with other “works of the flesh,” like covetousness, malice, rivalry, and resentment (Galatians 5:19–21).
But as tricky as envy is — in both its pervasiveness and hiddenness — it is highly susceptible to the sword of the Spirit (Ephesians 6:17; Romans 8:13). Awareness of envy’s masks, and direct confrontation through faith in the promises of God, go a long way in smiting the green-eyed monster. As does thankfulness. “It’s hard for envy to hide in a grateful heart,” says Rigney.
In this new episode of Theology Refresh, Rigney takes this bull by the horns, not only making us mindful of its power and patterns, but also preparing us to fight. He gets us ready not just to play defense against envy — “the great leveler,” as Dorothy Sayers called it — but also to go on the offensive in pursuit of the riches of God’s lavish inequality. That’s right, there’s a kind of inequality that God loves, and discovering the beauty of God’s gracious variety (Romans 12:6; 1 Corinthians 12:4; 1 Peter 4:10) just might be one of the breakthrough for you against envy.
To access this 13-minute episode, subscribe to Theology Refresh in iTunes, listen at the resource page, or download the audio.
Joe Rigney is professor at Bethlehem College & Seminary in Minneapolis. He gave the seminar “Live Like a Narnian” at the recent Desiring God 2013 National Conference, and is author of a new book by the same title, available in softcover and for Kindle.
Recent episodes of Theology Refresh:
To subscribe or see the full list of almost 50 episodes, visit Theology Refresh in the iTunes store.
The Church and the World: Homosexuality, Abortion, and Race with John Piper and Douglas Wilson [Desiring God Blog] 2013-10-04 10:30
All the hot-button topics were on the table Sunday night in downtown Minneapolis. Bethlehem College and Seminary hosted a dialogue on Christ and culture with John Piper and Douglas Wilson, moderated by Joe Rigney. The video is now available.
Early on, the conversation turned to slavery, racism, and Wilson’s controversial stance which sparked a lengthy online debate with Thabiti Anyabwile just months ago. Piper, who closely followed the entire debate, offered his seasoned reflections on the interchange.
Wilson shared about growing up in segregated Annapolis and how his father trained him to hate the discrimination. He also gave the backstory to his provocative book Black and Tan, and Piper offered six reasons for his ongoing association with Wilson — including, “Doug hates racism from the core of his gospel soul.”
The dialogue then transitioned to abortion, and later to homosexuality, its prominence in America, and how the Church can vocally oppose the pro-gay agenda without alienating Christians who struggle with same-sex attraction.
The full conversation is captured in this two-hour video — and see the detailed timestamps below for the bits you might find particularly interesting.
0:00:00 • Hellos, intros, and prayer.
0:04:02 • Should moral chaos in America make us panic?
0:09:15 • Should moral chaos in America make us passive?
0:16:06 • Douglas Wilson’s position on slavery and racial reconciliation.
0:21:10 • John Piper’s takeaways from Wilson and Thabiti Anyabwile’s 111-page blog discussion on slavery and race.
0:22:53 • Six reasons the risk of partnering with Wilson is worth it for Piper.
0:26:55 • Wilson on whether his presence jeopardizes racial reconciliation.
0:30:12 • Wilson on the Anyabwile debate.
0:31:57 • The backstory behind Wilson’s book Black and Tan.
0:36:42 • Piper on making emotional connections in reconciliation.
0:39:20 • Piper on growing up in segregated Greenville, SC.
0:40:38 • Wilson on growing up in segregated Annapolis, MD.
0:42:58 • Wilson: The key to all reconciliation is God-centeredness.
0:45:50 • Wilson: We need to receive hard truths from African American brothers, and be willing to offer them.
0:46:56 • Why do African American Christians vote for pro-abortion politicians?
0:54:48 • Abortion as the slavery of the 20th century.
0:58:26 • Is the slavery/abortion link insensitive?
1:03:02 • Do you care about your reputation in debates? And how should Christian leaders address controversies (bold offending, cowardly retreating, non-offending edification)?
1:16:13 • Where is our culture on homosexuality? Where is it headed?
1:17:48 • Wilson: The central problems of culture are worship problems.
1:23:02 • Piper: How to be an effective idol-blasting Christian.
1:24:33 • Piper: I don’t know where culture is going. Collapse? Revival?
1:26:01 • Wilson: God’s kingdom progresses from triumph to triumph via apparent disasters (the cross).
1:28:06 • Why Piper avoids sodomy language in the homosexual debate.
1:35:06 • Why Wilson uses sodomy language in the homosexual debate.
1:39:48 • How to attack cultural apostles of homosexuality without alienating Christians struggling with same-sex attractions.
1:45:22 • Piper: Be slow to judge the heart of a Christian speaking online.
1:47:35 • If persecution is coming to America, how should the Church ready herself?
1:53:26 • Piper closes in prayer.
God’s Bright Design for Your Bitter Providences [Desiring God Blog] 2013-10-04 01:00

This is the will of God, your sanctification. (1 Thessalonians 4:3)
God tells us everything we need to know to live godly lives (2 Peter 1:3). But sometimes we wonder.
The unexpected, unexplained twists and turns our lives take create all kinds of apparent uncertainties for us. And the profound pain we endure can be so perplexing. There is so much God doesn’t tell us — so much we think we would really like to know.
But as Deuteronomy 29:29 says, “The secret things belong to the Lord our God, but the things that are revealed belong to us and to our children forever.”
This means that as creatures we must learn to live contentedly with what God intends to be mysterious to us and grab hold of the revealed things with everything we have.
The secrets God keeps from us are a greater mercy to us than we likely realize. We often forget just how thin is the sliver of reality we see and information we can contain at any given time. Humans are not equipped to handle what the Bible calls “the knowledge of good and evil” (Genesis 2:17).
When we want God to start giving us some answers, we need the Bible to help us get our heads out of the claustrophobic confines of our private worlds and into the galactic greatness of what God is allowing us to be a part of. We need to remember that we’re dealing with God here.
God is a person for whom time, space, and matter present no limitations. He has dimensions accessible to him that we know nothing about. He is Trinitarian in his essence (Matthew 28:19). He holds tens (maybe hundreds) of billions of galaxies together by the word of his power (Hebrews 1:3). He has created and governs every throne, dominion, ruler or authority (Colossians 1:16) and every being that is invisible to us, whether angel (Romans 8:38) or demon (Luke 4:41). He is orchestrating all of human history (Acts 17:26) with its multiple billions of complex individuals past, present, and future — of which each of us is only one — and multiple trillions of interweaving causes and effects — of which each of us only experiences a relative handful. And he’s working all of these things toward a point when every knee will bow and every tongue will confess that Jesus Christ is Lord to the glory of God the Father (Philippians 2:10–11).
And we wonder why we struggle to understand what God is doing in our difficult circumstances.
God is doing ten thousand things in our circumstances! That’s likely a significant underestimate.
We would fall on our faces in awe-filled worship if we saw the chain reaction for our eternal good (Romans 8:28) and that of other present and future believers that God is engineering in just one seemingly random occurrence (Proverbs 16:33) that today might be the source of our grumbling because of the grief it is causing us. Now think of a lifetime’s worth.
God doesn’t explicitly promise this, but I tend to think that one of the glories and joys of the age to come will be God’s unveiling of the bright, extensive designs of his bitter providences in this age and the grace upon grace upon grace that they unleashed while we, not knowing, simply held on to Proverbs 3:5 with all our might.
But what God does explicitly promise is that every moment and level of suffering we experience as we live by faith (Romans 1:17) “is preparing for us an eternal weight of glory beyond all comparison” (2 Corinthians 4:17).
That thing that you don’t want, that you’re weary of, that you plead with God to remove (and might remove at some point unless he says otherwise [2 Corinthians 12:9]) is preparing you for glory.
God’s will for you is your sanctification (1 Thessalonians 4:3). He wants you to share his holiness (Hebrews 12:10). And the kindness of God in pursuing this for you is incomprehensively wonderful because without his holiness you will have no real and lasting happiness. Only in his presence is fullness of joy (Psalm 16:11) and only the pure in heart will see him (Matthew 5:8).
You want him to make you holy. You really do. Whatever it takes.
The God who governs the visible and invisible worlds knows what he is doing in your life. The God who was brutally murdered on a Roman cross knows what it’s like to suffer and how to redeem it. Specifically how he will bring good out of your trials may be mysterious to you now, but that he will bring good out of them is not a mystery. It’s a promise.
And this is where you get to participate with God in your sanctification! You get to act the miracle. You work out your salvation (Philippians 2:12) by believing the promises God makes to you (John 6:29).
And as you believe God’s promises you will bear the “peaceful fruit of righteousness” (Hebrews 12:11), which are the attitudes and actions of those who live by faith in the Son of God (Galatians 2:20). Behavior always follows belief. So your belief will result in faith-filled obedience to God (Romans 1:5) and produce various kinds of faith-fueled works for God (2 Thessalonians 1:11).
The secret things are the Lord’s for a very good reason. Trust him with the mystery. But the revealed things are yours and they are glorious. Believe them and one day you’ll share God’s holiness and all the forevermore pleasures he has prepared for you (Psalm 16:11).
Each of the headers in bold are lines from William Cowper’s beloved hymn, “God Moves In a Mysterious Way.” Acting the Miracle: God’s Work and Ours in the Mystery of Sanctification, edited by John Piper and David Mathis, with contributions from Kevin DeYoung, Ed Welch, Jarvis Williams, and Russell Moore, is now available in softcover and a free PDF.
Recent posts from Jon Bloom:
A Biblical Theology of Love [Desiring God Blog] 2013-10-03 11:17

Love is at the heart of the Bible.
God loved us so much he sent his only be-loved Son to love us by blood, so that we would likewise love and treasure this be-loved Son (John 3:16, Revelation 1:5).
But that doesn’t tell the full story. On the cross, Christ initiated a love to break our love-less sin, to gift us with new hearts, and to make us love-givers (1 John 4:19). The Holy Spirit pours God’s love into us (Romans 5:5; Galatians 5:22).
Such love has a full-bodied Trinitarian flavor, with ancient roots dug deep in the Old Testament. And one scholar building on this theme is Jason DeRouchie, Associate Professor of Old Testament at Bethlehem College and Seminary in Minneapolis.
DeRouchie is the editor of an innovative new book: What the Old Testament Authors Really Cared About: A Survey of Jesus’ Bible, a full-color, illustrated survey of the Old Testament storyline from a distinctly Christ-centered and Christian perspective (Luke 24:44).
DeRouchie penned the notes on Deuteronomy, a book he is well versed in, and I recently asked him for a mini biblical theology of love beginning with Deuteronomy and the famous Shema, which reads: “Hear, O Israel: The Lᴏʀᴅ our God, the Lᴏʀᴅ is one. You shall love the Lᴏʀᴅ your God with all your heart and with all your soul and with all your might” (Deuteronomy 6:4–5).
All love begins with God, DeRouchie told me, “Because in his call for us to love him, that is where we know greatest joy for eternity. God loves us by calling us to love him.” And it’s a call to an all-embracing love, to love him with our heart and soul — our desires, our will, and all our motivations.
“But bigger than that,” DeRouchie said, “it’s a love that includes all our actions, our words, how we treat others, our perception, everything connected with our being.” Our love for God is all-encompassing; it includes loving God with all our might (literally, all our ‘very-ness’), which includes all our powers, wealth, resources, everything: “our car, our wife, our social media, our clothing, our children, our house. Love for God is wholehearted, life-encompassing, and community-embracing. Everywhere we go, everything we do cries out: ‘The Lᴏʀᴅ is one in my life!’”
But such a love expressed from our lips and hearts and wallets and smartphones requires a radical heart transformation. This has always been true (Deuteronomy 10:16). To love God and to love our neighbors, like the Law commanded, requires new hearts. In the Old Testament, this is the promise of the coming New Covenant (Ezekiel 11:19–20, 36:26).
The gospel is essential for every eternal love. “All my love is a blood-bought love,” DeRouchie said. “My joy in Christ, my delight in all that God has secured for me, is now what defines me. I am in this world to magnify the greatness of God shown to me in love. And every decision I make, every encounter I have, is about loving God, which overflows in love of neighbor. The fuel for loving my neighbor in a self-sacrificial way, regardless of the cost, is my joy and satisfaction in God.”
But this is only a start. For more on how love develops in the storyline of the Bible, listen to our full 39-minute conversation. There we address some practical Christ-centered ways love gets refreshed in our lives, lessons drawn from 1 Timothy 1:5.
Catch our full conversation by subscribing to the Authors on the Line podcast in iTunes here, or download the mp3 here (26.9 MB), or listen in from the resource page through the following link:
A Biblical Theology of Love: An Interview with Jason DeRouchie (39 Minutes)
A sampling of previous Authors on the Line podcasts —
It’s Time to Celebrate [Desiring God Blog] 2013-10-03 01:00

“He heard music and dancing.”
This line introduces the first main action of the “older brother” in Jesus’s parable of the Prodigal Son. The rest of the story culminates with what he doesn’t do.
On his way home from the field, the older brother heard the commotion. “Music and dancing,” we’re told. And we’d expect, if there’s music and dancing, there’s laughter and cheers. This is a party. The father had ordered a celebration: “Let us eat and celebrate” (Luke 15:23). Which is what happened: “And they began to celebrate” (Luke 15:24).
It doesn’t take us readers long to see that this is a big deal. The word for “celebration” is used four times in this parable, and it means merriment! It was the kind of rejoicing that if found in other contexts for different motives it would be wrong, which is the case when this word is used two other times in Luke’s Gospel (Luke 12:19; 16:19).
But the older brother wants no part in this party. He got mad when he found out the news behind this noise. Literally, “he did not desire to enter” (Luke 15:28). He refused to join the joy. So his father came out to him and entreated him. The father pleaded, implored, urged his eldest son to share in his pleasure. But the eldest son still refused. He complained, twisting what the father had graciously done for his brother to be about what he had not done for him. He recoiled at the display of mercy and no doubt exposed several signs of corruption deep inside. But his father’s response simply called out the obvious . . .
The older brother’s problem was that he was not glad when he should have been.
“It was fitting,” the father says, “It was fitting to celebrate and be glad, for this your brother was dead, and is alive; he was lost, and is found” (Luke 15:32).
In the full picture of Luke’s Gospel this parable stands as an indictment against the unbelieving Jewish leaders. Jesus had launched a new day in redemptive history. The unfolding drama of God’s salvation was extending beyond the borders of ethnic Israel — even to “the highways and hedges” (Luke 14:23). The wayward sons and daughters from afar were being gathered. This was a promised day, a long-awaited day in the history of Israel and the world. This was the day of salvation — a day to celebrate which continues to the present (2 Corinthians 6:2). But the Jewish leaders in the midst of this day were not glad, and they were wrong. And if we are not glad now, the same goes for us.
God commands us to be joyful: “Delight yourself in the Lᴏʀᴅ” (Psalm 37:4); Rejoice in the Lord always (Philippians 4:4). The basis of this joy is God himself. He displays his manifold perfections and satisfies our deepest desire. “Come, everyone who thirsts!” he tells us (Isaiah 55:1). This alone is enough to necessitate our joy.
Now add to this the mighty works of God occurring at this particular epoch of human history. The Holy Spirit is on the move. The gospel is advancing to all nations. Jesus is building his church. It is fitting that we celebrate and be glad. And therefore, in a real sense, to the degree that we refuse to celebrate and be glad, we show ourselves to be out of touch with God’s triumph in this world. And if we are indifferent, if we prefer to huff and puff, we must be as clueless as the Pharisees.
To be clear, there is nothing shallow about this joy. The event that inspires this pleasure goes deeper than the superficial successes that tend to snag us. The point is not to celebrate and be glad because a reality TV show about Christians is topping the charts. Rather, this joy is a kind not of this world. It’s more like: our brothers and sisters in the Middle East are being killed, our buildings are being burned, our children are threatened, our witness marginalized, but the gates of hell will not prevail. Sinners are being saved.
The good news of Jesus, simultaneous to our sufferings, and often through them, is penetrating the darkest darkness all over this world. There is no depth his love cannot reach and no power that can stay his hand. Jesus is building his church. Whatever circumstances we find ourselves in, this is cause to rejoice.
The time is right. It is fitting to celebrate and be glad.
Recent posts from Jonathan Parnell:
Dieting, Botox, and Honoring Jesus with Your Body [Desiring God Blog] 2013-10-02 11:05

Undistracting attractiveness is John Piper’s term for it.
It’s a vision for the Christian to steer a middle course between idolizing our bodies and neglecting them. It includes giving our bodies enough attention — with sleep, diet, exercise, and upkeep — to avoid being distractingly unattractive, and reining in our impulses to pursue a self-focused attractiveness that distracts.
Your body is a precious gift from God. And the Bible is clear about its highest purpose: To make God look good.
Do you not know that your body is a temple of the Holy Spirit within you, whom you have from God? You are not your own, for you were bought with a price. So glorify God in your body. (1 Corinthians 6:19–20)
And so Paul writes, “It is my eager expectation and hope that I will not be at all ashamed, but that with full courage now as always Christ will be honored in my body, whether by life or by death” (Philippians 1:20).
Beneath the flashpoints of dieting and exercise, botox and plastic surgery, lies this question for the Christian: Are we using our bodies in such a way that God is seen to be more precious than our bodies?
“People can tell if you’re focused more on the inner person of beauty than the outer person of beauty,” says Piper. He points to 1 Peter 3:3–4, which was written for women, but also is applicable to men:
Do not let your adorning be external — the braiding of hair and the putting on of gold jewelry, or the clothing you wear — but let your adorning be the hidden person of the heart with the imperishable beauty of a gentle and quiet spirit, which in God’s sight is very precious.
C.S. Lewis called the body “Brother Ass.” It’s serviceable, and not too impressive, and we should be thankful when it does its job and gets us where we need to go. Like a loyal donkey.
And the best of bodies in this age will soon find their beauty frustratingly illusive. Says Piper, “The most voluptuous female body you have ever seen in your life is going to be ashen faced in a coffin before she knows it — and then where will be all the investments in that temporary, outward beauty?”
Today’s episode of Ask Pastor John also includes advice for singles who are especially eager to be fit and attractive to get attention from the opposite sex, as well as more on “present[ing] your bodies as a living sacrifice, holy and acceptable to God” (Romans 12:1).
Ask Pastor John now has surpassed 2 million plays since its inception earlier this year. Thank you, to all who have listened and submitted questions. Here are the three most played episodes released in the past month:
Ask Pastor John is a daily podcast series of 3–8 minute conversations released each weekday at 10:30am (EST) through the DG Facebook and Twitter feeds. You can tune in to the new episodes through the free Ask Pastor John mobile app for iPhone and Android. We’re currently hosting all the recordings on SoundCloud, a website making it easy to listen to several of the podcasts in one sitting. They’re also archived on the DG website and syndicated in iTunes.
We want to hear from you. To submit a question to Pastor John please include your first name, hometown, and question in an email to AskPastorJohn AT desiringGod DOT org.
Does God (Really) Desire All to Be Saved? [Desiring God Blog] 2013-10-02 01:30

On the extent of who will be saved, the Bible makes two clear points:
But how these two biblical truths (that seem to contradict) actually relate, has perplexed theologians and inquiring Christian minds for many centuries, sparking vigorous debates and (more recently) fiery comment threads on Facebook.
This pair of doctrines force questions like:
These are just a few of the thick questions involved.
Finding the answers is like climbing Mount Everest. Not everyone is up for the climb, but we believe it can be done, and there are guides to help if you want to make the attempt. John Piper offers himself as a Sherpa of sorts for the steep climb in his new little book, Does God Desire All to Be Saved?
If you’re asking these types of questions — and if you’re up for the climb — the 50-page book is available as a free download here, or purchase here.
Failure to Live on Mission Is a Worship Problem [Desiring God Blog] 2013-10-01 11:30

Sometimes we think the way to engage people in mission is to make sure we get the right information to them.
If we just preach the Bible, people will evangelize.
If we show people the commands in Scripture to care for the poor, people will develop a heart for mercy ministry.
If we make people aware of our need for more volunteers, people will sign up.
In other words, we perceive a knowledge problem. People need to know how to apply the Scriptures better, and once they know what they need to do, they’ll do it.
But this isn’t the way long-term change takes place. Most of the time, when we are marked by missional apathy, it’s not that we don’t know what we ought to be doing; it’s that we don’t want to be doing what we ought to be doing.
In our efforts to increase missional fervor, we can get so focused on giving people more information, or better application, that we forget that our main task is to lead people to exultation. That’s a fancy word for “worship.” We exult, we delight in the Savior, we revel in him. Exaltation of the Savior leads to exultation of the saints.
Lack of mission is rarely a knowledge problem; it’s a worship problem. We don’t have any trouble talking about the things we love most. Whenever we find something worthy of attention, we talk about it.
The same is true of our relationship with Christ. The more we are in awe of his worthiness, the more likely we are to speak of him to others and serve others in his name.
Sometimes people worry that the rough edges of Christianity will lead us to avoid serving our neighbor and sharing the gospel. So we play down some of the harder truths of the gospel, not denying them of course, but not giving them their proper weight.
The reality of hell is an example. There are all sorts of ways to downplay the truth of one’s eternal destiny; the most common is simply to not speak of it, or to recast salvation as dealing more with this life than the next.
But what happens when the reality of hell is no longer grounding our talk about salvation and the gospel? We miss out on a moment of worship.
Consider this scenario. You’re walking with a friend, not paying much attention to where you are headed. Suddenly, your friend grabs your arm and yanks you backwards. At first, you are annoyed that you’ve been stopped so suddenly. But then your friend points in front of you. Sure enough, he had a reason. You were about to step off into a ditch, where you might have broken your foot or sprained your ankle. Your annoyance turns to gratitude for his “saving you” from possible harm. You thank your friend and move on.
Consider the same scenario, except this time your friend doesn’t pull you back from a ditch, but a cliff. You were about to fall to your death, hundreds of feet below. What would your reaction be in this situation? Not just a word of “thanks.” You’d be crying and hugging your friend, overflowing with gratitude for the way he just saved your life.
In the same way, when we minimize the severity of God’s judgment for sin, we are less inclined to stand in awe of the marvelous salvation Christ has provided for us. We think we’re pushing aside an obstacle when we neglect the reality of judgment. But what we’re actually doing is pushing away one of the truths that most leads us to worship. The reality of God’s grace is all the more amazing the more we see our sin and what it deserves.
A gospel-centered teacher isn’t satisfied to see his people learn truths about God. A gospel-centered leader wants them to feel those truths. To feel the full weight of God’s provision for us in Christ. To have the heart’s affections stirred to worship the loving God who has saved us by his grace and incorporated us into his family.
Does “Mere Christianity” Mean Eliminate Denominations? [Desiring God Blog] 2013-10-01 01:00

For many years my conviction has been that Christian unity and Christian truth are served best not by removing fences, but by loving across them and having welcoming gates. I don’t claim to do it well. I want to do it better.
The point is that minimizing truth, or filing down its clear edges, or blending it all into one indistinguishable mass, or focusing on prayer, service, and mission, rather than truth — none of these produces unity that honors truth, creates robust communities, or endures for generations.
That happens best when we live well in our communities of conviction, and love well across convictional lines.
Would C.S. Lewis agree with this? Didn’t he write Mere Christianity? Doesn’t that imply we should lay aside our denominational differences and live in the visible unity of “mere Christianity?”
You may be surprised by what Lewis means by this phrase. But he tells us clearly. What follows is an excerpt (in italics) from the introduction to Mere Christianity (1943, xi–xii) broken into sections with my comments.
I hope no reader will suppose that “mere” Christianity is here put forward as an alternative to the creeds of the existing communions — as if a man could adopt it in preference to Congregationalism or Greek Orthodoxy or anything else.
When Lewis writes about mere Christianity he is not criticizing Christian denominations. In fact, he says it’s not as though a person even “could” make mere Christianity a standing place. It would be like saying that the shirt I wear is neither sleeveless, short-sleeved, or long-sleeved. It’s just a shirt.
[Mere Christianity] is more like a hall out of which doors open into several rooms. If I can bring anyone into that hall I shall have done what I attempted. But it is in the rooms, not in the hall, that there are fires and chairs and meals.
Lewis loved the Church of England. It was his denominational home. But he did not see his calling as an advocate for Anglicanism. His calling was to lead people into the hall of the house of Christianity. And he knew that the hall was not the place anyone should live.
This is the mistake many have made about Lewis. He was not ecumenical in the sense of leading people out of denominational rooms into the hall of unity. His ecumenical spirit will be seen below as love between rooms, not the emptying of rooms into the hall.
The denominational rooms are where the fire and chairs and meals are. In other words, if you try to live in the hall, you will go without warmth and rest and food. Mere Christianity is not lived Christianity. Trying to make it a life is like trying to eat mere food but never eating any particular vegetables or fruits or meats.
The hall is a place to wait in, a place from which to try the various doors, not a place to live in. For that purpose the worst of the rooms (whichever that may be) is, I think, preferable.
He is so clear about the inadequacy of mere Christianity that he says that living the best you can in the worst Christian denomination is better than trying to live in the hall.
It is true that some people may have to wait in the hall for a considerable time. . . . You must keep on praying for light: and, of course, even in the hall you must begin trying to obey the rules which are common to the whole house. And above all you must be asking which door is the true one; not which pleases you best by its paint and paneling. In plain language, the question should never be: “Do I like that kind of service?” but “Are these doctrines true: Is holiness here? Does my conscience move me towards this?”
This is one of the reasons I love Lewis. There is no gibberish here about all the rooms being equal. Or all the rooms having the same truth from different angles. Or personal experience being the main thing, while truth-claims are a human presumption. Or the inadequacy of saints to make good judgments about which denomination has truth. None of that.
No. Instead there is the straightforward statement that a crucial move must be made from the hall of mere Christianity to the doctrinal specificity of a room. To that end your primary task, once you are in the hall, is to discover the room closest to the truth. So he urges us to “keep on praying for light.” And to “ask which door is the true one.” And to inquire not whether we like the services but “Are these doctrines true?”
When you have reached your own room, be kind to those who have chosen different doors, and to those who are still in the hall. If they are wrong they need your prayers all the more; and if they are your enemies, then you are under orders to pray for them. That is one of the rules common to the whole house.
This is Lewis’s ecumenism. Choose a denominational room according to biblical truth as best you can. Then love those who choose differently, even if they turn out to be enemies.
What the world needs from the great house of Christianity is not that all the walls be knocked out between the rooms, but that we love each other in all the ways the Bible says, including defending and confirming the truth of Scripture as we see it (Ephesians 4:15).
Read this post in Arabic, Chinese, Portuguese, or Spanish.
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“Disunity in Christ”: An Interview with Christena Cleveland [Pure Church by Thabiti Anyabwile] 2013-10-31 03:39
Note: Christena Cleveland is a social psychologist with a hopeful passion for overcoming cultural divisions in groups. She regularly blogs about reconciliation, race and privilege. Drawing from a vast body of research, she uncovers the underlying processes that affect relationships within and between groups and helps leaders understand how to promote an appreciation for diversity and build effective collaborations with diverse groups. Christena earned a B.A. from Dartmouth College and a Ph.D. from the University of California. She has published numerous scholarly articles and held academic appointments at the University of California, Westmont College, St. Catherine University and Bethel Seminary. She coaches pastors and organizational leaders on multicultural issues and speaks regularly at organizations, churches, conferences and universities. In addition to speaking, coaching and writing, she serves on the pastoral preaching team at her church and is a volunteer Young Life leader in urban Minneapolis. She recently completed her first book Disunity in Christ: Uncovering the Hidden Forces that Keep Us Apart.
I had the privilege of offering an endorsement of Christena’s book, which I loved as a social scientist. Here’s my plug:
In Disunity in Christ, Christena Cleveland provides an insightful analysis of why we all say we want unity but find it so difficult to gain it. Combining a humble Christian tone, familiarity with many types of churches and skillful use of social science, Disunity in Christ reveals to us those very human tendencies that keep us divided. Along the way, Cleveland helps us to see, laugh at and rethink our very selves. This book will effectively help any Christian or church wanting a deeper experience of the reconciliation we have in the body of Christ. As a pastor serving a church of some thirty nationalities, I found it an extremely useful analysis of what hurts and helps unity.
Christena was kind enough to answer a few questions for Pure Church.
When and how did you first become interested in diversity issues? What has been the biggest influence on your thinking?
My mom claims that I was born interested in diversity and justice issues! She can tell you some crazy stories about me as a six-year-old, boycotting recess time at school because the kids running the school yard games excluded the blind girl in the class. I guess I’ve always noticed and valued difference and desired to bring everyone into the circle despite those differences.
My neighborhood was my first diversity lab, so to speak. I grew up in Fremont, California, the 2nd most diverse city in the United States. With over 9 nationalities represented by the kids of my block, I encountered cultural differences – from holidays and religious observances to food preferences to perceptions of time – every afternoon when we gathered to play kickball in the street.
When I was 8, my dad planted a church in our city – a church that became almost precisely 25% black, 25% white, 25% Asian, and 25% Hispanic. To my young mind, it made perfect sense that I would attend a multiethnic church in a multiethnic city. It was at this church that I began to get a vision for how God desires us to be in relationship with each other, despite cultural conflicts, theological differences and vastly varying worldviews.
The biggest influence on my thinking has been the Gospels. When I examine Jesus’ heart and actions, I see a consistent cross-cultural theme. It seems that everything Jesus did was cross-cultural: the Incarnation, his meaningful relationships with a diverse group of people, his ability to speak to people in a way that affirmed their specific culture, the Cross. By examining the Gospels, I’ve discovered that a significant part of following Jesus involves caring about people whose experiences, cultural backgrounds and problems are nothing like my own.
You begin the book with a delightful discussion of what you call “right Christian, wrong Christian.” What do you mean by that phrase and how does it affect unity in the local church?
I actually went back and forth on whether to include that discussion in the book! In that section, I show my cards, if you will. So, I was concerned that non-discerning readers who identified with my description of “Wrong Christian” would throw my book across the room and never pick it up again!
“Right Christian, Wrong Christian” is all about naming our biases and recognizing that many of us have so succumbed to the tribalism in the church that we’ve started labeling people who are like us as “right” and people who are different than us as “wrong.” The problem is that many of us have little ongoing, meaningful interaction with the people we’ve labeled “wrong Christian.” As a result, our perception of “wrong Christian” more closely resembles a caricature than an accurate and honoring portrait.
Meanwhile, our negative attitudes toward “wrong Christian” blind us to the fact that perhaps we’re not the “right Christians” that we think we are. I see this pattern of instinctively, unequivocally and judgmentally labeling other followers of Christ as wrong or right on a broad level (e.g., in the blogosphere – where cross-tribal engagement only happens when one person/group is protesting another person/group) and on a local level (e.g., in the local church – where individuals within local church bodies stick to people who are like them / agree with them and avoid meaningful interactions with those who are different / challenge their worldview).
Many people feel Evangelicalism has become increasingly “tribal” in recent years. It seems we’ve become adept at placing people in categories. Is this tendency to categorize helpful or unhelpful?
It’s both. As more diverse groups within Evangelicalism gain a voice and a distinct identity, it’s helpful to use categories to keep track of all of the groups! In that way, categories and labels are helpful. But the sinister side-effect is that by using those categories, we erect seemingly insurmountable divisions between us and them. Before long, we’re no longer thinking of them as a different but invaluable part of the same body of Christ. Instead, we’re thinking of them as wholly and categorically different than us – so different that they are now both wrong and invaluable. What starts out as a mere label to help us distinguish between the wide variety of groups in the body of Christ can easily morph into a monstrous divisions that makes us lose sight of the fact that the most important label is our common identity as Christians.
You write in the book, “The body of Christ is like a bad marriage.” Wow. What do you mean by that?
In my social psychology class, the students and I examine lots of research on satisfied and dissatisfied couples. Some of the most interesting findings show that dissatisfied couples assume the worst of each other, tend to discount positive behavior and tend to attribute negative behavior to global, stable causes like personality.
For example, if a wife in a distressed marriage wakes up early on Saturday to surprise her husband with breakfast in bed, he’s likely to interpret her positive behavior by saying, “She must want something from me.” Or, “She probably couldn’t sleep. She only made breakfast for me because she was bored and it gave her something to do.”
However, if the wife in a distressed marriage commits a negative behavior, say she forgets to tell him that she’s coming home late from work and will have to miss dinner, he’s likely to interpret her negative behavior by saying, “It’s because she’s a selfish person.” He’s unlikely to think that she’s an unselfish person who simply happened to forget to call this time.
So the husband disregards the wife’s positive behavior and assumes that her negative behavior is fueled by stable personality deficiencies. As you can see, the husband and wife never sit down to have a meaningful conversation. Instead, the husband’s perceptions of the wife are wholly based on his assumptions. In a distressed marriage like this, no matter what the wife does, she loses!
I’m sad to say that I see this dysfunctional pattern of relating in the body of Christ. People from different tribes often act like the disgruntled husband in the distressed marriage. We tend to zero in on the “negative” behaviors that other Christian groups are engaging in and we tend to attribute those behaviors to personality deficiencies (e.g., “They don’t value Scripture” or “They’ve become too worldly”). Meanwhile, we barely notice the positive things that other groups in the body of Christ are doing. If we notice them at all, we often assume that their motives are impure, that they have an “agenda” or that they’re not worth listening to because they’re outside our tribe.
What suggestions would you have for church leaders wanting to lead their churches into becoming more diverse and unified communities?
I think one of the most powerful things that church leaders can do to lead their churches into unity is to model unity in their own personal relationships. A church will never be diverse if the leaders don’t live diverse lives. Engaging in meaningful cross-cultural contact is scary! But people follow their leaders. If they see their leaders doing it, they will likely follow suit. Indeed, research shows that when a group leader models a relationship with a non-group member, the members of the group automatically begin to perceive the non-group member in a more positive way!
Church leaders should start by building meaningful friendships with people outside their ethnic, political, theological, gender, class, age, and marital status groups. When they begin to do this, they may begin to realize that the people who they had previously labeled as “wrong Christian” are now some of their most trusted friends. As part of this process, their misperceptions of them will begin to come to light.
The work of reconciliation is hard, slow, sometimes costly work. You’ve often encouraged people to persevere in the work and take the risks. Why do you think it’s worth it?
The work of reconciliation is the work of the Cross. I love doing it – despite the high costs – because it keeps me on my knees at the cross, asking our Savior to infuse me with his reconciling love so I can share it with others and participate as an empowered co-heir in His great work of making all things new. There really is no better place in the world to be!
Thanks for inviting me to interact with you and your readers! I love interacting with fellow members of the body of Christ and I’m excited to be on your blog today!
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FREE BOOK GIVE-AWAY
IVP is kind enough to offer five free copies of Christena’s book to readers of the blog. Here’s how it works. The first five people to tweet a link to this interview and plug the book will receive a free copy. When you tweet be sure to include my twitter handle (@thabitianyabwil) so I’ll know you’ve done so. I’ll connect with you to get your mailing details and IVP will send you Disunity in Christ. You’re going to enjoy this read!
“Disunity in Christ”: An Interview with Christena Cleveland
Ja Rule Exposes My Sinful Heart [Pure Church by Thabiti Anyabwile] 2013-10-23 03:20

This week Marc Lamont Hill of HuffPost Live interviewed rapper Ja Rule about life after a two-year prison sentence, his new movie, “I’m in Love with a Church Girl” and his newfound faith. Much of the hip hop community has been abuzz with the news of Ja’s faith. For those who haven’t seen it, here’s the full 19-minute segment.
00:50 Introduction to his new movie, “I’m in Love with a Church Girl”
02:00 Parallels between the movie’s main character and his own life
04:50 How he feels about his career right now?
05:40 Perspective on his prison experience
07:30 Talks about his new music projects
09:10 Relationship with 50 Cent and obligation to be a role model
12:10 Was he ahead of his time?
13:23 His new relationship with God
15:38 First impression of Hillsong Church (NYC)
18:20 What he teaches his sons and daughter as privileged children
When a young brother at the church asked if I’d seen the interview, I quietly suspected Ja Rule’s profession might be like a long line of incredible testimonies by celebrities looking to turn over a new leaf. I confess: I’m somewhat jaded by awards shows and interviews featuring artists whose work glamorizes sin while they claim to know God. From Al Green’s on-again-off-again relationship with R&B and gospel to Kanye West’s “I’m a Christian,” I find it difficult not to be skeptical.
But as I watched Hill’s interview with Ja Rule, I found a number of things working in my heart that it’s better to confess than suppress. I kept blinking at some logs and thought I’d share a few.
1. I realized I didn’t know and wasn’t interested to know Ja Rule. More than likely I heard his music and with mild disdain or self-righteousness turned it off and blamed him for destroying the community.
2. I never prayed for Ja. His conversion has nothing to do with my petitions for I never petitioned for his conversion. I’m aware of how often I’ve used my lungs to complain about and criticize secular hip hop and how little I’ve used my lungs to cry to God for the men and women involved in it.
3. I was struck by the surprising reversals and cunning providence of God. Ja said when he was poor he was sure he would go to jail; then when he was successful he was sure he would never go to jail. In His rich mercy, God spared him jail when he had nothing to lose and sent him to prison when he had everything to lose. The ways of God are not my ways; His thoughts are far higher than my own. “He who observes providence will have providences to observe.” (Flavel)
4. Legalistic religion “puts a black eye” on the gospel. I was surprised to learn Ja grew up a Jehovah’s Witness. I was saddened to hear, once again, how deeply bruised and resistant legalism left him. The Law kills. Yet I suspect my inner Pharisee makes himself known more often than I’m aware.
5. I’m tempted to not celebrate the work of grace in a sinner’s life. I’m tempted to doubt their testimony and conversion. I’m like those members of the early church who heard of Paul’s conversion and responded with more than a little apprehension and doubt.
6. I’m tempted to doubt the power of the gospel to save. Even though I know and I preach the ability of the gospel to save terrorists. Why would I doubt that a man like Ja Rule (which is to say, a man like every other man in sin) would not be saved when they came under the sound of the gospel?
7. I’m tempted to judge the methods of other churches and to doubt God’s use of them. I hear “Hillsong” and I squint that suspicious disapproving squint. As if God’s hand is shortened that He can’t reach people in a service unlike the service I think is “right” or at least “more faithful.” By the way, I know nothing about Hillsong. There’s that Pharisee again.
8. I might even be mildly disappointed with God for using Hillsong rather than a “solid church.” I’ve got my own Jonah thing going on.
9. Sometimes it takes radical difference to awaken people to the power of the gospel.
10. The authenticity of the church continues to be a significant stumbling block for some outside the church. Does the church really mean “come as you are”?
11. I sometimes underestimate the ability of multi-ethnic churches to reach people steeped in mono-ethnic subcultures. There’s a little homogeneous unit principle assumption sneaking around in my thoughts, especially when it comes to cats steeped in hip hop culture. I would never have put Ja in a Hillsong church. But God did. And praise be to God that’s where He chose to work!
So Ja Rule exposed me. And I’m glad. Now I’m happy for him and I’m praying for Him. I’m praying for Hillsong and the preaching of God’s word there and the fellowship of God’s people. May they be used of the Lord to bring many sons to glory through the preaching of the cross of Christ.
Ja Rule Exposes My Sinful Heart
“Art: In the World but Not of It”: A Conversation on Christian Hip Hop and Culture [Pure Church by Thabiti Anyabwile] 2013-10-22 03:21
I’ve been waiting for audio or video of the recent Unashamed conference to be released. Like a lot of people, I wanted to be there, and wanted to hear shai linne and Sho Baraka discuss the current thinking about faithful witness and cultural engagement in Christian hip hop circles. I’ve heard nothing but good things about the panel they shared, moderated by J’son.
As I’ve watched this debate, I’ve been moved to pray often for the brothers in this endeavor. I’ve been challenged to think and re-think some positions of my own. And I’ve been encouraged to see the manful efforts at preserving the unity of God’s people where differences of strategy exists. I think these videos go a long way in continuing that godly path set out by brothers engaged in the discussion.
If you’ve not listened in before, feel free to check it out below.
“Art: In the World but Not of It”: A Conversation on Christian Hip Hop and Culture
“Words in Season”: An Interview with Leon Brown about Live and His New Book on Evangelism [Pure Church by Thabiti Anyabwile] 2013-10-21 03:13
Editor’s Note: This year I’ve been thinking about evangelism and try to grow as an evangelist. I’m no great evangelist, so I appreciate those who are more faithful and effective than I am. Leon Brown is one such brother. He’s written a new book on evangelism entitled Words in Season. You can learn more at the book’s website, where you can download a free pdf of the Introduction and chapter 1. You can also purchase the book from Amazon. The book boasts a wonderful set of endorsements. It has been a pleasure to grab Leon for a few interview questions about his life and his book
1. Tell us about yourself? How did you come to faith in Christ? Was an evangelist instrumental in your conversion?
Perhaps my story is a bit atypical in reformed circles. Please know as I share these things, I’m not attempting to be melodramatic; it’s simply my story.
My only exposure to “church” prior to coming to Christ was a Mormon “church.” That isn’t necessarily the atypical part. I was born a crack baby, raised in a single-parent household, frequently watched my family members imbibe illegal substances, gun shots in the neighbor were commonplace. I dropped out of high school my sophomore year—I had to in order to survive; my mother abandoned me when I was fifteen. A couple of years later, I received a General Education Diploma (GED). At eighteen, I joined the Navy.

Although I have many good childhood memories (e.g., the conversations I had with my mother, the friendships I developed, etc.), the aforementioned experiences greatly shaped me. Interestingly enough, I wouldn’t reorder one moment of my life, especially now that I’ve come to recognize God ordains all that comes to pass.
Thankfully, there is a story much bigger than my own—a story that provided great hope for a young African American that should have been claimed by the negative statistics surrounding ghetto youth.
Around the year 2000, a retiring Navy serviceman invited me to church. There, I was introduced to the wonders of God in Christ. There, God saved me. And now, as a pastor in the Presbyterian Church in America (PCA), I live to make Christ known. God has also granted me the opportunity to continue my education. Currently, I am working toward my Ph.D. in Classical and Ancient Near Eastern Studies. I’m also married to my lovely wife, Rosalinda. We have one daughter, Genesis Rose.
2. How did you become involved in evangelism? Was it something you began to do right away, or was there a pivotal moment where you caught a passion for it?
Like the woman at the well, telling people about Jesus was the natural byproduct of my introduction to him. Initially, I didn’t know all of the more in depth particulars of the gospel (e.g., why Jesus was born of a virgin, how did each person of the Trinity take part in the plan of salvation, etc.), but the simple request to unbelievers to, “Come see a man!” was sufficient. Then in time as I grew in the Lord, I was able to express the gospel more clearly and if needed, talk about some of the more difficult concepts of the salvation message. To date, however, I’m still learning; I’m still a work in progress.
3. Why did you write this book?
Words in Season was birthed out of a desire to see people encouraged about the sharing the gospel and inviting people to church. Far too often I think many people feel like they have to be the next Billy Graham or major evangelist. With such a weight on their shoulders, it can sometimes be difficult to get excited about talking about Christ and his Church. If you’re so worried about filling the shoes of the next man, you won’t ever get comfortable in your own shoes. So in writing this book, I wanted to help people see that God made them in unique ways; he provided them with certain gifts; and they can utilize those gifts in sharing the good news of Christ to the glory of God.
4. Who is the book for?
Overall, I had three groups of people in mind. I wanted to encourage those who struggle with sharing their faith. I wanted them to know that it’s possible and that the Lord can use even the most timid person. Secondly, I wrote this book for those who presently enjoy sharing their faith, but they don’t recognize the importance of the local church. This latter group sometimes forms “evangelism cliques” and verbally assaults the church because people don’t seem as exited as those within the clique to witness of Christ. (For a time, I belonged to this group). Thirdly, I wrote this book for those who don’t believe laypersons are commanded to share the gospel. While the chapter titled, “Must I Really Share My Faith?” is brief, I hope that the scripture references provided at least furthers the discussion on layperson witnessing.
5. What do you think is key in becoming a motivated and effective evangelist?
In order to become a motivated and effective evangelist, I think we should keep several things in mind. 1) We share our faith for the glory of God. 2) We must remain bathed in grace. 3) It’s not about us. 4) God’s word is always effective (Isa. 55:11). 5) Well, you’ll have to read the book to find out more.
6. There are a number of books on the market. What makes this book different?
There are many good books on personal evangelism. I’m thankful for the many faithful Christians who’ve sought to put their ideas on paper. Having said that, I wanted to write a book that didn’t seek to teach a paradigm. While I give tips on sharing the gospel, I tried to develop a concept that exhorted Christians to be who they are while sharing the gospel. In other words, don’t be like me—be you! I don’t want people subscribing to a formula. As I point out in my book, that can come across formulaic and a bit too mechanical. Rather, I encourage people to listen to those to whom they are talking; love the unbeliever because he are made in the image and after the likeness of God; even show hospitality to the unbeliever. Also, at the end of every chapter, I’ve inserted discussion questions. I wanted small groups and churches to continue the conversation. In fact, I wrote the book to be used in small group Bible studies and Sunday school. Lastly, I wanted to highlight the importance of the church as a gospel-driven organism. The Church is a witness! Every Lord’s Day, the Church testifies both to Christians and an unbelieving world. I don’t think we should highlight the importance of individual witness to the exclusion of the Church. Therefore, chapter two is about the Church!
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For a bonus, here’s Leon on a Modern Reformation interview discussing how to respond to objections to the Christian faith in evangelistic conversations:
“Words in Season”: An Interview with Leon Brown about Live and His New Book on Evangelism
Why You Should Care about the “Strange Fire” Discussion [Pure Church by Thabiti Anyabwile] 2013-10-18 03:19

Well, the “Strange Fire” conference is underway. The twittersphere is lit up like a Christmas tree and the partisan battle lines are drawn deeply in the theological sand. As far as I can tell, you’re likely to fall into one of four positions:
1. Cessationist
2. Continuationist
3. Saddened
4. Could care less
I understand the first three categories. If you’re in group 1 or 2, you likely have biblical reasons for why you are. Hopefully you’ve wrestled with the biblical text, the well-formed thoughts of others–both pro and con, and you’ve landed as best you can on what you think is biblical ground.
I understand the third group, too. You may be in either group, but you’re mainly dejected at the sight of Christian leaders you respect “going at each other” over a vitally important but secondary issue. You’re wondering why it has to be this way. You’re feeling more and more like Rodney King. It’s not that you think Christians can’t or shouldn’t disagree. You perhaps feel Christians shouldn’t disagree this way. Not your heroes.
It seems to me the last group has the weakest position. “I could care less” and “let’s move on already” can’t really be justified by any of the Bible’s teaching. After all, what’s really being debated is how we walk with God. If we could care less about that, then we couldn’t care less about God himself. So this post is really a simple plea to folks tempted toward category 4 to care more. Think more. Feel more. Listen more. Give attention to this debate because in doing so you’ll be giving attention to the ways of God, how you might know Him better, how you might keep in step with His Spirit, and how you might discover the joy of fellowship with Him. I can’t think of a more important topic.
But as we give attention to these presentations and responses of various sorts, it seems we really must keep a few things in mind. Things that, to me at least, indicate that this is no light or laughing matter. An honest discussion on this topic entails a number of uncomfortable admissions. And, perhaps it’s our inability or unwillingness to admit these things that hinder our discussion as much as anything else. We feel the stakes but we don’t want to face the stakes. For some people, this conference forces some difficult admissions, admissions we’d be happier not to concede. But, that, too, is reason to care about the issue and engage prayerfully. Consider what’s at stake.
First, we have to admit that there’s a correct and an incorrect position on this issue. Somebody is right and somebody is wrong. The outcomes are non-correspondent. The thing can’t be “A” and “not A” at the same time in the same way. Those who are wrong are teaching error. That error impacts the next two things we need to admit.
Second, we have to admit that how we view this issue substantially impacts the nature of the Christian life. It matters. It’s not an inconsequential idea. Someone worships God appropriately, someone doesn’t. Someone walks with God in a way that pleases Him, someone doesn’t. Our view of these things informs our personal communion with God.
Third, we have to admit that this issue practically impacts Christian worship and fellowship. It’s not only a private matter, but a corporate one as well. If we want to apply what we think the Bible teaches regarding gifts, it’s going to have a material impact on who we can actually worship with. It’s like baptism. We will either limit it to professing believers or we will include covenant children. But we can’t do both. Our decision about practicing or not practicing some secondary-but-important issues affects who can belong to our churches and what we’ll do when we gather. We may continue personal friendships (and we should), but we’ll find it difficult to continue corporate fellowship.
Fourth, we have to admit the Bible does not answer the issue compellingly. Or, better, in our fallen reading of the Bible someone–perhaps everyone–is not understanding and applying what we ought. From what I can tell, everyone in this discussion believes the Bible is sufficient for matters of doctrine and devotion. I see people of varying perspectives affirming that. But if the Bible were as clear to everyone as we’d like, we wouldn’t be having the conversation. So, it seems we’ve got to interrogate ourselves about (a) whether we’re reading the Bible with a squint, such that things that ought to be seen lose focus, and/or (b) whether the Bible really intends to tell us all the things we desire on this topic. I believe the Bible is sufficient in all that it says, but perhaps people across the spectrum are tempted to make it say more than it does say.
I read one tweet where a brother called for more “epistemic humility.” That’s a good phrase, I think. We might also call for some more “exegetical humility.” But “epistemic” or “exegetical humility” ought not be confused with “I could care less.” The two are miles apart. One receives God’s revelation as it really is, the word of God; the other waves an unconcerned hand in the face of God’s word and the godly men attempting to hold forth the truth. One takes God’s word seriously; the other tends toward a practical atheism that shuts His voice out. Always better to seek the truth of God’s word, even if we see dimly, than to swear off God’s word and debates about it.
So, whether you’ve watched the live feeds or not, whether you’ve read multiple volumes on the debate or not, I hope you care about these things. I hope you’re following with careful concern to know the truth rather than to vindicate your party. I hope you’re listening with rapt attention because you’re eager to hear God’s voice more clearly and to walk with your Savior more closely. Even the thoughts of folks who get some things wrong can help us to do that if we’re discerning and humble beneath God’s word.
May the Lord bless His Church.
Why You Should Care about the “Strange Fire” Discussion
Christ Is Not Ashamed to Call Us “Brothers,” Therefore Be Unashamed [Pure Church by Thabiti Anyabwile] 2013-10-16 03:47
I love this promo video for T4G 2014. I pray you will join us there. And, more importantly than that, I hope we’ll all pray to be unashamed at every opportunity we have to make Jesus known!
Christ Is Not Ashamed to Call Us “Brothers,” Therefore Be Unashamed
Black Parenting on Prime Time Television Through the Years [Pure Church by Thabiti Anyabwile] 2013-10-14 03:09
One of the dearest brothers in the Lord to me had his first taste of African-American parenting culture when his son was the only white player on an inner-city Pop Warner football team. He told me about how he marveled at how loud everyone was. The coach seemed to be always yelling, and yet there was never any doubt about his love for the kids.
When my wife first began teaching high school social sciences, she often found herself coming to aid of African-American students who were being written up and chastised by her white colleagues (usually female). Most often the kids were in the hall bustin’ on each other. For many of Kristie’s peers it was a cross-cultural experience they didn’t know how to interpret. Occasionally they were correct; something more serious was in the mix. But very often they mistook a good round of crackin’ for actual hostility. In truth, knowing how to crack back earned you some measure of respect.
Here are a few of my favorite moments from prime time television. They each give a brief glimpse into the kind of parenting I grew up with–full of authority, sometimes quite emotional, always loving. I like these snippets because they capture some core lessons that African-American parents of my generation passed along to us kids. Now, if this seems foreign to you and perhaps a little harsh, I want you to keep something in mind (especially before you comment). Racial prejudice and discrimination was/is the fierce background against which African-American parenting occurred. The aim of so much Black parenting was to prepare our children for a world that would be a thousand times more fierce, unforgiving, and deadly than anything a parent could say in these situations. If a parent seemed to spit out a rebuke, keep in mind their children were likely to be spit on by others. If the parent threatened physical punishment, keep in mind the child was entering a world that sometimes threatened physical death. Now, I certainly don’t condone child abuse. Nor do I think these scenes amount to abuse. In some of them, it’s how you love and instruct when stakes are higher than you can communicate to your child. With that, enjoy!
Clair v. Elvin (The Cosby Show)–Or, the best of family life includes mutual respect and service.
Twice as Good (Scandal)–One of the clearest messages sent to kids in my era: The racial prejudice of the culture does not mean you can’t achieve; it means you got to be twice as good. It ain’t fair, but it is what it is and you better show up.
Michael Gets Suspended (Good Times)–For the longest times, African-American parents placed great stress on getting an education. Long before “acting white,” there was “get an education.” And students of promise could expect some of the sternest discipline for wasting a chance at an education.
I Brought You in This World, and I’ll Take You Out (The Cosby Show)–Had to end with another from King Cos. Lesson: Don’t question my authority.
Black Parenting on Prime Time Television Through the Years
They Said It Far Better Than I Could [Pure Church by Thabiti Anyabwile] 2013-10-10 11:20
Recently I’ve attempted to argue that in our discourse about homosexuality we need to return the discussion to the basic description of the acts themselves. I’ve suggested that on two grounds, one fairly implicit, the other stated explicitly. Implicit in my previous posts was the assumption that the entire premise of homosexuality as social identity needs to be questioned. I didn’t develop this thought, but it was working in my description of how the public conversation about homosexuality turned so quickly and decisively. The more explicit statement was that we need to turn the conversation to the sex acts themselves because the success of the pro-homosexuality campaign depends on our not considering those things actively.
This week a couple of pieces make those points far more eloquently and helpfully than I could ever do.
Understanding the Perception and Rhetoric
The first comes from a New Yorker profile of Edith Windsor, the plaintiff in the DOMA case. At one point in the interview, the discussion turns toward rhetorical strategy and public perception. Here’s the relevant bit:
When selecting the ideal plaintiff, one experienced movement attorney told me, “Women are better than men, post-sexual is better than young.” From the Bible onward, two men having intercourse has been viewed as more disturbing to the social order than two women doing whatever it is that lesbians do. For people to embrace same-sex marriage, they needed to focus on the universal desire for romantic love and committed intimacy. Contemplating the difference between gay people and straight people made it acceptable to treat their relationships unequally, and the difference between homosexuality and heterosexuality is sexuality. Provided that Kaplan kept her client muzzled on the topic, Americans could imagine that Edie Windsor had aged out of carnality.
This interview accomplishes in a paragraph what I clumsily tried to illustrate by retelling that private public policy discussion from a decade ago. The ability to make homosexuality an accepted practice in the minds of the mainstream public depends upon a public presentation of homosexuality as effectively “aged out of carnality.” Or to put it another way, it depends on dulling the conscience by avoiding those behaviors widely rejected by the public. Unless we understand that this is the intentional, active strategy of one side (and I don’t blame them one bit for making their best case; it’s what we all do!), then we won’t be engaged in an honest conversation. Those who oppose certain laws that enshrine homosexuality as “good and right” (like so-called “gay marriage”) will continue to joust the windmill of public perception rigged by a political presentation of the case. Here you have the rhetorical strategy described in the words of those supportive of gay rights. Opponents would do well to adjust accordingly.
Questioning the Very Construct of Homosexuality
Of course, the most fundamental form of this discussion requires we consider the concept of homosexuality itself. What was once known as sodomy and universally regarded a sin has become a “sexual orientation” considered a product of nature by many. Many people would like to call that debate “closed” and consider the matter settled firmly on the side of nature rather than nurture.
First Things posted a thoughtful and engaging piece today entitled, “Sexual Disorientation: The Trouble with Talking about ‘Gayness’.” Michael Hannon asks, “perhaps it is worth asking whether the premises themselves, and the formal framework in which they operate, should not be rejected wholesale. I wonder in particular whether employing the concept “gay people” with such nonchalance may communicate a familiarity and friendliness with this concept that is unmerited by its pedigree.” The entire piece is worth reading, but here are the salient points regarding how we think and talk about homosexuality.
1. “In his Histoire de la Sexualité, Michel Foucault argues that homosexuality is a social construct, and one constructed terribly recently at that.”
2. “Of course, that homosexuality is a social construct does not automatically render it evil or necessitate our rejection of it.”
3. “Still, while social constructs may be often benign, and may be sometimes even beneficial or necessary, there is good reason to doubt that sexual orientation is such a constructive construct.” Hannon elaborates:
First of all, the heterosexuality-homosexuality distinction is a construct that is dishonest about its identity as a construct, masquerading as it does as a natural categorization, applicable to all people in all times and places according to the typical objects of their sexual desires (albeit with perhaps a few more menu items on offer for the more politically correct categorizers). Claiming to be not simply an accidental nineteenth-century invention but a timeless truth about human sexual nature, this framework puts on airs, deceiving those who adopt its distinctions into believing that they are worth far more than they really are.
A second reason to doubt whether this concept is one that we Christians should readily employ is that its introduction into our sexual discourse has not noticeably increased the virtues—intellectual or moral—of those who utilize it. On the contrary, it has bred both intellectual obscurity and moral disarray. Our young people, for instance, now regularly find themselves agonizing over their sexual identity, navel-gazing in an attempt to discern their place in this allegedly natural framework of orientations. Such obsessions invite far more heat than light, and focus our already sexually excited adolescents on discerning extraneous dimensions of their own sexual makeup. This becomes thornier yet for those who discern in themselves a “homosexual orientation,” as they adopt an identity distinguished essentially by a set of genital sexual desires that cannot morally be fulfilled.
Again, the entire piece is worth reading and considering. Its most helpful virtue is its attempt to put us back on first things (appropriate for the magazine!), to help us ask the most basic questions lest we continue to find ourselves swept along by the winds of political spin, careening toward experiments and ideas contrary to historical facts, nature, and revelation.
They Said It Far Better Than I Could
Christmas in the Mailbox: Some New Books to Read [Pure Church by Thabiti Anyabwile] 2013-10-03 10:49
One of the great joys of coming home from a month’s travel is seeing what’s in the mailbox. Especially those wonderful publisher envelopes and boxes bearing books! And having been away for a month, there was a hefty collection of envelopes waiting to be opened and books to be appreciated.
Here are a few that arrived that I hope to dig into in the months ahead. Perhaps some of them will be a blessing to you.
Kevin DeYoung, Crazy Busy: A (Mercifully) Short Book about a (Really) Big Problem (Crossway)
I plan on starting with this book, as soon as I find time! I need this book, and I needed it to be as brief as it is. With Kevin’s engaging and creative writing style I’m sure this will not only be a practical help to my life but also the kind of book I’ll want to read repeatedly.
T. Desmond Alexander, From Eden to the New Jerusalem: An Introduction to Biblical Theology (Kregel)
We need short biblical theologies for people new to the discipline. Skimming the pages it seems Alexander has managed to combine two things that rarely co-exist: brevity and readability as an introduction with scholarship reflected in a ton of footnotes for those who want to chase down weightier works. I look forward to reading through From Eden to the New Jerusalem because I’d love to have an updated entry-level biblical theology to pass along to new and growing believers.
David Platt, Daniel L. Akin, and Tony Merida, Exalting Jesus in 1 & 2 Timothy and Titus (Holman)
This is the inaugural volume in B&H’s new expositional commentary series, Christ-Centered Exposition. The series editors happen to be this volumes authors: Platt, Akin and Merida. These three brothers love God’s word, take it seriously in preaching and living, and provide excellent models of exposition. It’ll be my privilege to contribute to this series with a text on Luke. If you’re looking to expand your library of expositional commentaries or to learn from faithful teachers like Platt, Akin and Merida, take a look at this volume.
D.A. Carson (ed), The Scriptures Testify About Me: Jesus and the Gospel in the Old Testament (Crossway)
A couple years ago The Gospel Coalition’s biennial conference focused on biblical theology. To preach Christ from the Old Testament, TGC invited Al Mohler, Tim Keller, Alistair Begg, James MacDonald, Conrad Mbewe, Matt Chandler, Mike Bullmore and D.A. Carson to preach from various genres and books of the Old Testament. Now those addresses are available in this concise volume tracing the storyline of the Bible.
Jon Bloom, Not by Sight: A Fresh Look at Old Stores of Walking by Faith (Crossway)
Jon Bloom is cofounder and president of Desiring God. He’s invested a considerable portion of his life helping others seek their highest happiness in Jesus Christ. For those who love stories, this collection of short sketches of biblical figures will surely encourage and strengthen the saints. Bloom aims to help us understand what it means to walk by faith, “following the unseen into an unknown, and believing Jesus’ words over and against the threats we see or the fears we feel.”
Paul David Tripp, What to Did You Expect? Redeeming the Realities of Marriage (Crossway)
So I finally got my copy of this! Yeah! I don’t know how this has escaped my possession and, more importantly, my reading for so long. If you know Paul Tripp then you know this is a searching, biblical, challenging and ultimately redemptive look at “the realities of marriage.” With a glut of books on the market that purport to tell us how our marriages “can be better,” we desperately need books that deal with our marriages as they truly are. I look forward to benefiting from this in my own marriage and as I counsel other couples.
Thomas R. Schreiner, The King in His Beauty: A Biblical Theology of the Old and New Testaments (Baker)
For scholarship and usefulness, you can’t get much better than Schreiner. I find him lucid, balanced and careful with the biblical text and our contemporary contexts. I’m looking at this 646 page theological overview of the Bible and I’m thinking, (1) I love the title of this book and I think it’s pages will help me see Jesus’ beauty and (2) I need to take a few other Schreiner titles off the shelf and have me a long period of Schreiner studies.
Anthony L. Chute, Christopher W. Morgan, and Robert A. Peterson (eds), Why We Belong: Evangelical Unity and Denominational Diversity (Crossway)
Boasting endorsements from Packer, Noll, George O. Wood, Paul House, Stephen Nichols and others, this volume looks to tackle an age old problem–unity in diversity. How can we reflect a unity more robust than our local churches and denominations without undermining our denominational commitments. Like Carl Trueman, I’m glad denominations exist because “they indicate that somebody somewhere still believes something.” In this volume, a number of evangelical leaders and thinkers tell us why they hold to their denominational distinctives and why they consider themselves evangelicals. Contributors include Gerald L. Bray (Anglican), Timothy F. George (Baptist), Douglas A. Sweeney (Lutheran), Timothy C. Tennent (Methodist), Byron D. Klaus (Pentecostal) and Bryan Chapell (Presbyterian). Should be a good read.
John MacArthur, Strange Fire: The Danger of Offending the Holy Spirit with Counterfeit Worship (Thomas Nelson)
In a day and age when many people establish their theological positions based on the persuasiveness of personalities rather than the plain meaning of Scripture, it’s necessary that we hear prophetic declarations of both warning and truth. In a day where confessional and denominational loyalties appear to have given away to affinity networks and broad coalitions (of which I am a part and support), we need clarion calls to remember the important things about which we also differ and why. John MacArthur has often played that role in the last two generations. He adds another contribution sure to anger some, cheer others, and if read carefully instruct all. The book includes three sections, Confronting a Counterfeit Revival, Exposing the Counterfeit Gifts, and Rediscovering the Spirit’s True Work. I’m looking forward to benefiting from this book and seeing the fruit from the accompanying conference.
Chris Lassiter, You’re Grounded: Rooted in Truth in a Shallow World (Moody)
Chris Lassiter is a sports and lifestyle writer in Virginia and contributes to music industry publications like VIBE, HipHopDX.com, and Rapzilla. He’s served with Young Life for the last decade and serves part-time on his local church’s staff. He’s a churchman with an eye on the culture, particularly the urban and hip hop subcultures. He writes here with the hope of moving that subculture and this generation from shallow appropriations of Jesus Christ to a deep and rooted walk with Him. In 18 short, readable chapters, Lassiter takes us through Jesus’ identity, His relationship to sinners, spiritual disciplines, and membership in the local church. This looks like a perfect tool for discipling young people in the church, youth groups, and youth evangelism.
Jared C. Wilson, The Pastor’s Justification: Applying the Work of Christ in Your Life and Ministry (Crossway)
Okay. I need this book. Just the chapter titles yell, “Stop and read me, Thabiti!” Maybe they call your name, too. “The Free Pastor.” “The Holy Pastor.” “The Humble Pastor.” “The Confident Pastor.” “The Watchful Pastor.” “The Justified Pastor.” And that’s just the first section. The second section’s five chapters look equally inviting. This one is high on my list.
K. Scott Oliphint, Covenantal Apologetics: Principles and Practice in Defense of Our Faith (Crossway)
Here’s an introduction to Reformed apologetics in the tradition of Cornelius Van Til. The book has four pages of impressive endorsements and, most importantly, the contents look to cover important theological and practical ground.
Quay Hanna, Bus America: Revelation of a Redneck (Middle Relief Publishing)
This is the most interesting title I received. Quay was kind enough to send me the book after reading some exchanges on my blog. He wanted to encourage me with the story of God’s power in His life. Just receiving the book was an encouragement. I look forward to reading Quay’s story and marveling afresh at that secret hand of God which makes hearts new.
Christmas in the Mailbox: Some New Books to Read
Government Shutdowns and Church Budgets [Pure Church by Thabiti Anyabwile] 2013-10-02 10:35

Well, my weekend in Washington, D.C. was fulled with food, fellowship and a lot of fun. While catching up with friends, preaching at CHBC, and doing a little work for The Front Porch, the Federal Government crept toward and entered a shutdown by failing to pass a budget. At issue was Obamacare. At work were the same old partisan politics and clashes in political vision.
I watched with slight interest, until a question came to me. I wonder how many local churches approach shut down each year because of church factions, rival visions, and unclear stewardship priorities? I suspect there are many.
While the Federal Government was shutting down, First Baptist Church was coming to the end of its budget approval process. Like a lot of churches, our budget seasons force the elders and congregation to consider its priorities and face its constraints. And like a lot of churches, we sometimes find ourselves having to negotiate what feel like competing priorities with limited resources. It can be a tense time.
Though we’ve never been able to do everything we’d like to do or able to satisfy every group hoping for a change of some sort, by God’s grace, we’ve had budget seasons that have been peaceful, unifying and engaging. Over the years, I’ve tried to teach the congregation a few things about our budget that I hope preserve unity and strengthen our focus. I thought I’d pass them along in case they help others.
Don’t Let the People Forget That It’s All about the Gospel and Its Advance
It’s very easy to enter discussions about the numbers or the resources, about real cuts and changes that affect people, and forget the Good News. In fact, I think this is perhaps the most serious mistake a church can make. For once people forget Jesus and His saving work, they lose sight of why the church exists. So, it’s useful to present and discuss the budget as a document embodying gospel priorities. It’s useful to talk in terms of gospel and kingdom investments, perhaps organizing the report in a way that shows how the congregation’s giving gets distributed into gospel ventures (in church ministries, in the community, over seas, etc.). But keep the people’s eyes on Jesus.
Don’t Let the People Forget They’re One Family
Once people lose sight of Jesus and the gospel, they also tend to forget they’re a family, united to each other, and on the same team. They forget to extend grace to others as they turn to their “camps” or individual desires. It’s helpful to use the family language of the Bible rather than the business language of the corporate world. It’s helpful to create a familial tone–hopefully a tone that’s regularly used throughout the year. We try to liken our church budget to the budgets of our families. Just as we hopefully balance our budgets at home, live within our means, and focus on major family obligations, so we do the same in the church family. And just as our biological families stumble and wobble if the family isn’t on one accord about the budget, so our church will pull apart at the seams if the church family doesn’t remain on one accord. We’re a family. We stand and fall together.
Don’t Let the People Forget the Budget Is Just a Tool
God’s word is forever settled in heaven; church budgets are not. Budgets are simply plans. They’re not chiseled into granite. With an eraser or a push of the “backspace” button, budgets can be edited and recalculated. They represent our best thinking at the moment. As things change, we should feel the freedom to come back to some initiatives or initiate new ones during the year. The congregation should know it’s not passing some inflexible law but adopting an operating plan. Moreover, that plan is simply one financial way of defining our unity. The approved budget is the church’s tool for saying, “This is what we purposed to do with God’s blessing.”
Don’t Let the People Forget Their Responsibility
Frankly, I’m not very good at this. I hate talking about giving and money and budgets. I have to work to remind myself of the first principle–it’s about Jesus and the gospel. Then I’m able to champion the priorities in the budget, not the budget itself. I feel more comfortable keeping budget data before the people when I’m keeping Jesus before my own eyes. Then all that’s left is to connect the average member to our gospel priorities. We do that by turning our top line income projection into a per household or per member figure. This year we turned our projected budget increase into a per member per month giving increase. People have a difficult time with understanding seven-digit figures. We’ve never handled or seen that kind of money at one time. But they have a good sense of whether they can give another $100 per month or so. They know what that means for their discretionary spending, entertainment choices, groceries and the like. As the leaders call them to commit to a new budget, they’re able to think about whether they can make such a commitment and how to respond to the leaders’ proposal.
Don’t Let the People Forget to Pray
Nothing happens without prayer. Nothing extraordinary for the kingdom anyway. Conflict happens without prayer. Division happens without prayer. Harsh words and hurt feelings happen without prayer. Worldly wisdom and the flesh creep in without prayer. So, it’s important to pray. From the beginning of budget season, through the many reports and committee meetings, during the vote and on into implementation, we need to pray for the priorities and investments represented in the budget tool. Don’t just pray when you miss an important budget target. Pray before you know the numbers and especially pray when God blesses the giving of the church so you don’t take God for granted “when you enter the land.” Use the budget to encourage the people in prayer.
I pray that the Lord’s churches would not be shut down because His leaders and His people can’t agree on priorities. I pray the need to make Jesus known would be so deeply understood and embraced by all God’s people that budgets take their vastly secondary and utilitarian place. May our churches know great unity of purpose in Christ each budget season.
Government Shutdowns and Church Budgets
Spiritual Formation in Emerging Adulthood [Reformation21] 2013-10-28 22:17
David P. Setran and Chris A. Kiesling, Spiritual Formation in Emerging Adulthood: A Practical Theology for College and Young Adult Ministry. Grand Rapids, Baker Academic, 2013. v + 280pp. Paperback $21.99.Gravity [Reformation21] 2013-10-22 13:11
Spoiler alert: This review details key scenes in the movie and its ending.
The Glorious Groan of the Gospel [Reformation21] 2013-10-21 09:41
The total amount of suffering per year in the natural world is beyond all decent contemplation...In a universe of blind physical forces and genetic replication, some people are going to get hurt, other people are going to get lucky, and you won't find any rhyme or reason in it, nor any justice. The universe that we observe has precisely the properties we should expect if there is, at bottom, no design, no purpose, no evil, no good, nothing but pitiless indifference.
Recovering Classic Evangelicalism [Reformation21] 2013-10-14 14:40
Recovering What Exactly? A Review of Gregory Thornbury's Recovering Classic Evangelicalism: Applying the Wisdom and Vision of Carl F. H. HenryOnce upon a time, evangelicalism was a countercultural upstart movement. Positioned in between mainline denominational liberalism and reactionary fundamentalism, the evangelicals saw themselves as evangelists to all of culture. Billy Graham was reaching the masses with his Crusades, Francis Schaeffer was reaching artists and university students at L'Abri, Larry Norman was recording Jesus music on the secular record labels and touring with Janis Joplin and the Doors, and Carl F. H. Henry was reaching the intellectuals through Christianity Today. It was 'classic evangelicalism'.
Although he is certainly right to raise the question of Henry's hermeneutics, Vanhoozer seems disinterested in Henry's fundamental concern in the context of his argument in GRA: if one makes the author's intent supreme, and if one says the author's intention was a genre other than historical and scientific accuracy, we have opened up a Pandora's box. Once you make this move, Henry warns, you can take any problematic or disputed text in Scripture as a matter of genre confusion. As we will discuss later in this volume, this is precisely the interpretive move behind crucial abandonments of inerrancy in contemporary evangelicalism ... (pp. 106-107).
Vanhoozer makes a good point that Henry was so focused on maintaining evangelical affirmations that he downplayed the richness of language and its setting in canonical linguistic form. Vanhoozer is also correct that language does more theologically than Henry allows. But after revisiting the first seven theses set forth in volume 2 of God, Revelation and Authority, I am not sure that it does less. Henry reminds us that the content of any theological system flows directly from divine prerogatives. The material, expression, and agenda of doctrine either conform to this pattern or set sail into uncertain anthropological and sociological waters (p. 114).
If he were alive today, Henry would likely survey the steady stream of formerly Protestant thinkers and writers converting to Roman Catholicism, and while disagreeing with their theological reasons for leaving, would probably sigh and quietly say to the evangelical community who had lost them, "I saw all of this coming. You can't say I didn't tell you so." The converts are leaving for a milieu with a robust epistemology, a church convinced of its own doctrinal heritage, and a community not accustomed to so much self-criticism or so many second glances about things like truth, marriage, and what ultimately is the basis for human flourishing (p. 206).
The Importance of the Printing Press for the Protestant Reformation, Part Two [Reformation21] 2013-10-14 09:14
almost fifty identifiable printers of Luther's works in the 1520s printing in twelve separate locations...There are another seventy printers in various locations printing mostly Reformation tracts. Overall for the sixteenth-century, there are three hundred and ninety-one printers, eight hundred and ninety-four authors and one hundred and twenty-five cities....Eighty-two of the smaller locations where printers lived and worked have not been the subject of specific print research. The odds are overwhelmingly in favor of the contention that if a German printer published pamphlets especially in the 1520s, he published Protestant materials. What is often thought of as a war of pamphlets between the followers of Luther and the pope in Rome may be seen as a lopsided one.(5)
I have received the second and third parts of my Sermon on Confession from you and the first part from Melanchthon. I cannot say how sorry and disgusted I am with the printing. I wish I had sent nothing in German, because they print it so poorly, carelessly, and confusedly, to say nothing of bad types and paper. John the printer is always the same old Johnny. Please do not let him print any of my German Homilies, but return them for me to send elsewhere. What is the use of my working so hard if the errors in the printed books give occasion to other publishers to make them still worse?... I shall forward no more until I learn that these sordid mercenaries care less for their profits than for the public. Such printers seem to think: "It is enough for me to get the money; let the readers look out for the matter."(23)
The Importance of the Printing Press for the Protestant Reformation, Part One [Reformation21] 2013-10-07 14:32
... consisted of two stout upright pieces of wood joined by two horizontal beams. A screw, working in the upper beam and turned by a long bar, exerted pressure downward upon a wooden plank placed on the paper.... The pitch of the screw was made [steep] ... so that the necessary rise and fall was gained within the quarter turn obtainable with a fixed bar. The twisting motion of the turning screw was counteracted by suspending the platen (the wooden plank) from a hollow wooden box that slid inside closely fitting guides while the screw turned freely within. The upright beams were frequently braced to the ceiling to keep the press steady.(4)
Mind and Cosmos [Reformation21] 2013-09-30 12:13
Thomas Nagel. Mind and Cosmos: Why the Materialist Neo-Darwinian Conception of Nature is Almost Certainly False. Oxford: Oxford University Press, 2012. 130 pp. Hardcover: $24.95.Serial Choice? Hermeneutics and a High View of the Bible" [Reformation21] 2013-09-30 09:59
Covenantal Apologetics [Reformation21] 2013-09-25 09:29
K. Scott Oliphint, Covenantal Apologetics: Principles and Practice in Defense of Our Faith. Wheaton, IL: Crossway, 2013, 277pp. $19.99So, at a minimum, we have to recognize that there is no intrinsic or essential incompatibility between properties that God has necessarily and the essential properties of creation, even of human beings... God was able to bring them both together - to unify them - without violating any of the respective properties. Any notion of compatibility will have to allow that if this is true, then there is no incompatibility between God's character and the character of human beings. God can unite them both into one without merging or changing either (p.185).
Nor the Heart of Man Imagined [Reformation21] 2013-09-23 08:57
None of the rulers of this age understood this, for if they had, they would not have crucified the Lord of glory. But, as it is written, "What no eye has seen, nor ear heard, nor the heart of man imagined, what God has prepared for those who love him"-- these things God has revealed to us through the Spirit. For the Spirit searches everything, even the depths of God" (1 Cor. 2:8-10).
God is Impassible and Impassioned [Reformation21] 2013-09-16 12:07
Rob Lister, God is Impassible and Impassioned: Toward a Theology of Divine Emotion. Wheaton: Crossway, 2013, 333 pp. $22.99.Harriet Beecher Stowe's Theological Transition [Reformation21] 2013-09-16 09:37
a numerous class in the third generation of Massachusetts clergy, commonly called Arminian, - men in whom this insensible change had been wrought from the sharply defined and pronounced Calvinism of the early fathers. They were mostly scholarly, quiet men, of calm and philosophic temperament, who, having from infancy walked in all the traditions of a virtuous and pious education, and passed from grade to grade of their progress with irreproachable quiet and decorum, came to regard the spiritual struggles and conflicts, the wrestlings and tears, the fastings and temptations of their ancestors with a secret skepticism, - to dwell on moralities, virtues, and decorums, rather than on those soul-stirring spiritual mysteries which still stood forth unquestioned and uncontradicted in their confessions of faith. (10:6)
never expected to find truth agreeable. Nothing in their experience of life had ever prepared them to think it would be so. Their investigations were made with the courage of the man who hopes little, but determines to know the worst of his affairs. They wanted no smoke of incense to blind them, and no soft opiates of pictures and music to lull them; for what they were after was truth, and not happiness, and they valued duty far higher than enjoyment. The underlying foundation of life, therefore, in New England, was one of profound, unutterable, and therefore unuttered, melancholy, which regarded human existence itself as a ghastly risk, and, in the case of the vast majority of human beings, an inconceivable misfortune. (10:421)
"Wal," said Sam, leaning over the fire, with his long, bony hands alternately raised to catch the warmth, and then dropped with an utter laxness, when the warmth become too pronounced, "Parson Simpson's a smart man; but, I tell ye, it's kind o' discouragin'. Why, he said our state and condition by natur' was just like this. We was clear down in a well fifty feet deep, and the sides all round nothin' but glare ice; but we was under immediate obligations to get out, cause we was free, voluntary agents. But nobody ever had got out, and nobody would, unless the Lord reached down and took 'em. And whether he would or not nobody could tell; it was all sovereignty. He said there wa'n't one in a hundred, - not one in a thousand, - not one in ten thousand, - that would be saved. Lordy massy, says I to myself, ef that's so, they're any of 'em welcome to my chance. And so I kin o'ris up and come out, 'cause I'd got a pretty long walk home, and I wanted to go round by South Pond, and inquire about Aunt Sally Morse's toothache." (10:83-84)
Of course, as a Calvinist, he found food for abundant discourse in reconciling this absolute freedom of man with those declarations in the standards of the Church which assert the absolute government of God over all his creatures and all their actions. But the cheerfulness and vigor with which he drove and interpreted and hammered in the most contradictory statements, when they came in the way of his favorite ideas, was really quite inspiring. (11:59)
An Introduction to German Pietism [Reformation21] 2013-09-09 14:22
Douglas H. Shantz, An Introduction to German Pietism: Protestant Renewal at the Dawn of Modern Europe. Baltimore, MD: Johns Hopkins University Press, 2013, 520 pp. $32.00Our Make-Believe Parents: When Adam Becomes More Fiction than Fact [Reformation21] 2013-09-09 11:35
For we did not follow cleverly devised myths when we made known to you the power and coming of our Lord Jesus Christ, but we were eyewitnesses of his majesty. For when he received honor and glory from God the Father, and the voice was borne to him by the Majestic Glory, "This is my beloved Son, with whom I am well pleased," we ourselves heard this very voice borne from heaven, for we were with him on the holy mountain. And we have the prophetic word more fully confirmed, to which you will do well to pay attention as to a lamp shining in a dark place, until the day dawns and the morning star rises in your hearts, knowing this first of all, that no prophecy of Scripture comes from someone's own interpretation. For no prophecy was ever produced by the will of man, but men spoke from God as they were carried along by the Holy Spirit (2 Pet. 1:16-21)
I am astonished that you are so quickly deserting him who called you in the grace of Christ and are turning to a different gospel--not that there is another one, but there are some who trouble you and want to distort the gospel of Christ. But even if we or an angel from heaven should preach to you a gospel contrary to the one we preached to you, let him be accursed. As we have said before, so now I say again: If anyone is preaching to you a gospel contrary to the one you received, let him be accursed. (Gal. 1:6-9)
Thus it is written, "The first man Adam became a living being"; the last Adam became a life-giving spirit. But it is not the spiritual that is first but the natural, and then the spiritual. The first man was from the earth, a man of dust; the second man is from heaven. As was the man of dust, so also are those who are of the dust, and as is the man of heaven, so also are those who are of heaven. Justas we have borne the image of the man of dust, we shall also bear the image of the man of heaven. (1 Cor. 15: 45-49)
This is now the second letter that I am writing to you, beloved. In both of them I am stirring up your sincere mind by way of reminder, that you should remember the predictions of the holy prophets and the commandment of the Lord and Savior through your apostles, knowing this first of all, that scoffers will come in the last days with scoffing, following their own sinful desires. They will say, "Where is the promise of his coming? For ever since the fathers fell asleep, all things are continuing as they were from the beginning of creation." For they deliberately overlook this fact, that the heavens existed long ago, and the earth was formed out of water and through water by the word of God, and that by means of these the world that then existed was deluged with water and perished. But by the same word the heavens and earth that now exist are stored up for fire, being kept until the day of judgment and destruction of the ungodly. (2 Pet. 3:1-7)
Should Christians Love Their Country? [Reformation21] 2013-09-02 17:48
While our 'two kingdoms' citizenship always warrants biblical reflection, the situation facing Christians in America today demands special wisdom and grace. Throughout our lifetime, there have been two competing Americas, one theistic and the other atheistic; one which more or less respects our Christian heritage and upholds general biblical values, and one which aggressively opposes biblical morality and a godly outlook on life... With the assistance of a radically motivated media establishment, pagan America has made steady gains during each of the last several decades.
We must continue to love our country. Nothing would more please or serve the radical secularists than for Christians to jettison their patriotic impulses.We must joyfully love our neighbors and serve our communities, including those who vigorously oppose us in the culture war... By all means, there must be no violence, personal abuse, or vindictive spirit in the Christian defense of our national heritage. Our calling as Christian Americans is to 'overcome evil with good' (Rom 12:21), even if our virtues are slanderously labeled as hate.
I feel uneasy when I read that Christians should love this country, or any other country for that matter... I don't see anywhere in Scripture which calls me, or anyone for that matter, to 'love our country'America, like every other Western nation has had a remarkable yet chequered history - morally, economically and militarily. What are we to love, and what kind of love are we to show?
'Loving one's country' strikes me as a peculiarly American, and American Christian, thing to say. American patriotism has long been the slave to a rather romantic view of American history... Has America really ever been a Christian country? Is there indeed such a thing in the new covenant era? If there were, one might, as a Christian, be able to stretch the term 'love' for one's country. (emphasis added)In a Christian country we might expect there to be a higher standard of public conduct. We might expect public and private morality championed and a measure of spiritual depth and progress. A Christian may well be able to love such a country. Yet is this the case with America? (emphasis added)
Or are we talking about loving the people of America? Christian love aside, do we really love the mix of people in the States? The history of race relations in America sets the standard for no country, to be sure. Again, I want to emphasize that I am...just highlighting serious obstacles to the Christian in fulfilling Rick's counsel to love one's country.
Loving something is a very powerful idea. Love is defined and shaped after God's love for us; indeed, we do not know love outside of God's love. God is love himself and any human love, whether believing or unbelieving, follows God in this respect. The Biblical pattern for one's relationship to his country seems more aimed at respect and honor, than love. In rendering to Caesar what belongs to him, we submit to God's will in that He put in place the powers that be. Yet love does not seem to enter into this paradigm...I simply don't see in Scripture that the Christian is called to love his country. Yes, he is to submit, yield obedience, give honor, even die for one's country in armed conflict. But love I do not see.
Pay to all what is owed to them: taxes to whom taxes are owed, revenue to whom revenue is owed, respect to whom respect is owed, honor to whom honor is owed. Owe no one anything, except to love each other; for the one who loves another has fulfilled the law.
The Christian traveller (Jeremy Walker) [Reformation21 Blog] 2013-10-31 14:13
While immensely thankful for the benefits of modern travel, there are elements of it that are not in the first rank of Walker enjoyments. I tend toward dislike of the experience of being herded and managed, with even the temperature of the environment sometimes being adjusted in order to prompt appropriate dispositions. And there are, of course, those elements of being in confined spaces with a bundle of other sinners which tend to prompt more carnal reactions.
And so it was with that combination of weariness and amusement that I surveyed the departure lounge at Newark airport a few days ago on my way home from a delightful time of fellowship and ministry. All human life, if not quite there, was certainly well on the way to being healthily represented. Looking about me, I was struck by the prominent ways and means in words and in deeds by which various of my fellow wanderers were proclaiming their personal identity and spiritual allegiance.
There were Orthodox Jews, the coats and hats and hair raising their flags of affiliation. There were flamboyant metrosexuals, all pastel shades and skinny jeans and overcooked poses. There were the Disgruntled, those sour-faced regular travellers who can predict - and do, to anyone who makes eye contact - all that will be slow or go wrong with frightening accuracy. There were Hindu ladies, their dress and make-up speaking of their affiliations. Sikhs and Muslims rubbed shoulders in their religious uniforms. There were the Angry, like the chap who uttered a string of distinctly audible curses for a good ten or fifteen minutes after being subjected to a patdown, making sure that we all know that we are in the presence of Those Not To Be Messed With. There were the extravagant homosexuals, all loud giggles and shouty comments, hyper-camping for the benefit of those around them. Here are the Nervous, who do not know where to go or what to do, agitated and antsy, asking everyone the same questions repeatedly. Over there are the languid Rich, dressed up to the nines, oozing through the crowds and the barriers when the call goes out for the privileged few who get to enter the flying can ten minutes or so before the rest of us. Make way, too, for the harried and active Rich, in their well-cut suits and with their high-end luggage, rushing from their last lucre-producing meeting to their next one, and trampling all who are in their path. Over there is that decorated beast, the Tattooed Brit, looking for all the world like a thug of the first water, but possibly one of the most pleasant and cheerful individuals who will board the pla . . . no, my mistake, it was the thug version. Watch out for the Gorgeous Woman, who has gone to more effort for this flight than most would for their wedding days, dressed and manipulated from head to toe to catch the male eye. There is our New Age Friend, burdened by weighty beads and floaty veils, rainbow hues no doubt fending off all manner of ethereal bad news. You begin to wonder what the social media footprint of the gathering might be, as heat and hunger and the passing of time begin to prompt increasing agitation, what vapid online meanderings or noxious electronic effluent rises from the horde as we sit and wait.
All of which fascinating tableau left me asking, "By what means should I, as a Christian traveller, communicate my personal identity and spiritual allegiance?" I could, as a start, do some airport-lounge preaching, but I am not sure that it is the right environment, and the polite though armed gentlemen in the smart white shirts and blue trousers tend to look down on that kind of thing. The age-old device of carrying a Bible larger than a cabin bag is trickier in these days of the electronic reading device. To the casual observer, I would imagine I don't look that much different to most of the other reasonably-dressed male travellers (I add that little adjectival qualification for those of you who don't realise how much I can charge the general public not to see me wearing skinny jeans), and the same would be true of most Christians, I imagine. The prominent wearing of crosses is not my thing, neither would I normally go down the emblazoned garment line, as if "by their T-shirts you shall know them." Conversations of deliberately-penetrating volume with a fellow-believer are contrived, as would be kneeling for prayer or praying out loud (too much like the Pharisee on the street corner). And then there's the question of social media comment: is it a Christian response to offer a stream of bilious bleatings or caustic comments on the situation and its participants?
I wonder, though, if the starting point ought to be character, attitude translating into action. It might not immediately declare you to be a true disciple, it might only open the door to speak to one or two people, but it should be the bedrock of our testimony. In the mix with all the variety of my fellow-passengers, and regardless of their identities and affiliations, am I marked out by patience in the face of provocations, cheerfulness despite difficulties, politeness in the experience of frustrations, thankfulness in the receipt of blessings, responsiveness when entreated, helpfulness around the overwhelmed or incompetent, self-forgetfulness in the atmosphere of entitlement, peacemaking among the argumentative, kindness around the selfish, candour among the sniping, calmness in the face of danger, self-control in a place of indulgence, graciousness among the godless, and even prayerfulness when confronted by needs and concerns? If I have the opportunity - if, perhaps, by these means I win the opportunity - am I then equally forthright, simple, clear and winsome in explaining, as the Lord grants opportunity, my attachment to the Lord Christ?
There is no flamboyance here, no extravagant or overblown trumpeting of one's Christian identity. However, there may and should be a real communication of a genuine and distinctive spirit of one who is following after Christ Jesus. Do those who spend time with us under these and other such circumstances come away not simply with a sense of our niceness (although that may be part of it) but of a character elevated by something more substantial than the fancies of the world or the claims of false religion?
So, the next time you face a journey by plane, train or automobile (other modes of transport are available) and anticipate a prolonged period in close company with your fellow mortals, perhaps it would be worth asking yourself whether or not your demeanour, disposition and deeds will leave those with whom you have come into contact with a savour of Christ. We should cultivate a personal identity so rooted in him and a spiritual affiliation so governed by him that, if people know his name, there might at least be some sense in which they might take notice of us, that we have been with Jesus.
New Alliance Website (Gabriel Fluhrer) [Reformation21 Blog] 2013-10-31 11:48
No doubt, gentle reader, you have thought to yourself, "Where can I read more Reformed articles and find great Reformed resources on the web?" Well, the Alliance of Confessing Evangelicals is happy to answer that question with the launch of Place for Truth. Now, in addition to the Reformed musings here at reformation21.org, you can enjoy more excellent content at PFT. This new website provides thoughtful yet accessible articles ranging over biblical theology, systematic theology, church history, and practical theology emphasizing the continual need for the church to maintain the gains of the Protestant Reformation. Head over there now to read part one of David Wells' article "Why We (Still) Need Reform".
Effective personal evangelism: prayer (Jeremy Walker) [Reformation21 Blog] 2013-10-30 06:40
The marks of effective personal evangelism we have surveyed so far are love, tenacity, boldness, consistency and understanding.
The sixth mark of the effective personal evangelist is prayer. The place of prayer in this list is not a marker of its relative insignificance. Could it be that one of the reasons why, with our children, friends, colleagues and communities, we are less effective than we wish to be is because we have not proved to be men and women of earnest, pleading prayer, borne of a love for God that seeks his glory above all else and a love for people that longs to see them saved from sin? Think, for example, of our Lord's mourning over Jerusalem: "O Jerusalem, Jerusalem, the one who kills the prophets and stones those who are sent to her! How often I wanted to gather your children together, as a hen gathers her brood under her wings, but you were not willing!" (Lk 13.34). I know that this is not a prayer per se, but can we imagine that a spirit such as this would not find an outlet in prayers to his heavenly Father? Or consider the words of the psalmist: "He who continually goes forth weeping, bearing seed for sowing, shall doubtless come again with rejoicing, bringing his sheaves with him" (Ps 126.6) - is that merely generic and diffuse weeping, or does it not suggest some earnest and heartfelt pleading? Do you think that Paul taught men and women night and day with tears, and that there was no counterpart in his private wrestlings with God for a blessing on his labours? How often does the apostle assure Christ's people of his continued prayers for them, and how much would he have prayed for them to come to new life? He agonises until they are brought to birth and then to see them mature in the faith. We must be pleaders with God. Perhaps you think of the vast number of people to be reached, the great number of streets to be visited, the endless number of words that might be spoken, and you would be tempted to conclude that we do not have time to waste in prayer. I know one gifted man who would give the first hour of his appointed time in this work to poring over an open Bible and pleading with the Lord for a blessing on his efforts before he ever opened his mouth to men. Could it be that our relative prayerlessness lies behind both our faint appetite for the work and our feeble strength in it?
Halloween or the Lord's Day? (Rob Ventura) [Reformation21 Blog] 2013-10-29 11:31
Michael Ives, pastor of the Presbyterian Reformed Church of East Greenwich, RI, wrote a helpful piece on Halloween that is well worth reading. With the day fast approaching, this thought provoking article will assist us as we seek to live as Christians in this fallen and wayward world.
The article can be seen here:
A Plea to Pastors and Elders (Aimee Byrd) [Reformation21 Blog] 2013-10-28 15:40
I recently heard of yet another field experiment demonstrating how
easily a stranger at the park can lure an unsuspecting child. Even
though parents felt confident in their discussions about not talking to
strangers, and especially not to follow them anywhere no matter what,
the old, "I've lost my puppy, will you help me find him" ditty still
works like a charm. These faux abduction investigations reveal the
inadequacies of the whole "stranger danger" message. The problem is that
predators are very friendly; they don't look like the monsters that
their parents make them out to be. What child wouldn't want to help a
smiley guy with a picture find man's best friend?
There seems to
be a similar scenario going on in the world of "Christian" books. I'm
sure pastors are thrilled when they have congregants who love to read.
But what are they reading, and how are they processing the material?
The
other day I was in the middle of a workout when the phone rang. I
glance over to notice that it is my grandma. Better get that. As I push
the pause button and catch my breath to answer, grandma is spilling
praise over the phone to me about my newly released book. Of course my
grandmother is going to be biased, but it was encouraging nonetheless.
Just as I was getting excited about using my book to have deeper
theological discussion with my sweet grandma, she drops a bomb:
"However, I haven't finished your book yet because I've also been
reading another fantastic book called, Jesus Calling. Have you heard of
it?" Why yes, yes I have.
Immediately I began to wonder, how can
someone read the claims in my book alongside of the claims in Sarah
Young's Jesus Calling and say they are both wonderful? They say two
completely different things about how God speaks to us and conveys
Christ to us. I wondered how you could faithfully attend a Southern
Baptist church for most of eighty-four years and not have the
discernment skills to see when the sufficiency of Scripture and
authority of God's Word is threatened.
Meanwhile, as I'm still
talking, I hear a text message come in. When I get off the phone, I
check to see I received a message from my mother-in-law who is a Roman
Catholic. She wanted to encourage me that her friend from church was so
"blown away" by my book that she was buying copies for her daughters to
read. As I was happy to hear that, again I was perplexed. It seems like
reading without discernment isn't only a problem for Protestants. How
could a devout Roman Catholic, who brings communion to the shut-ins,
love what I had to say about things like justification, the church, and
the means of grace? I thought of the irony of being interrupted from a
physical fitness workout to be reminded of the need for theological
fitness.
I do a book review club at my house every now and then
where a group of women get together to review what we've been reading.
Even in this intimate setting with people who know a bit more of my
passion for theology, I've had women bringing in books such as Todd
Burpo's Heaven is For Real, and Rob Bell's Love Wins, with positive
reviews. These are popular books that attempt to persuade readers to
think differently from what the Bible says about heaven, the extent of
God's love, sin, and hell. I always get to play the role of the
unloving, bad guy when I ask more challenging questions about the books.
And
so now I make my plea. Pastors, elders, is there something more we
could be doing in the church to equip the congregation with discerning
reading skills? Good, Christian people are being deceived. Sadly,
sometimes it is those in leadership who recommend such books. The
so-called Christian author can actually be a potential predator of
orthodoxy in the church. They have great personalities and wonderful
stories to share, but where are they leading your flock?
Of
course, you shouldn't ban church members from reading such books. I can
tell my son to NEVER go anywhere with someone we have not pre-approved.
But how can you better provide the skills to recognize the ol' puppy
trick when it comes to reading choices? Maybe it is by actually reading
some of these books together, and teaching how to examine them against
Scripture. Maybe it is by teaching that niceness is not the same as
godliness. Perhaps we need to be challenged with our own hearts and why
we want to believe a message that is contradictory to the Word of God.
Whatever the case, the sheep are susceptible to looking for candy in
poisonous books. It's time to raise awareness for the church's need to
"learn to discern."
Big enough to write your own rules (Todd Pruitt) [Reformation21 Blog] 2013-10-28 10:48
It is not an easy thing to be the pastor of a mega-church. There are peculiar pressures and temptations that accompany such a role. Not least is the temptation to believe that you are big enough to write your own rules. The church's "success" is so intricately woven to your own that you become too big to fail. That is a dangerous place for any pastor and church. Add to that great sums of money and gratuitous praise from followers and the results are toxic.
More can be written about a story out of Charlotte, NC about a pastor and his great big house but suffice it to say that I now have another reason to be grateful for Presbyterianism.
Now, before my congregational brothers and sisters misunderstand, I am not saying that you are bad. I am not saying that autonomous congregationalism inevitably leads to the current outrage in Charlotte. It surely does not. I am saying that any well ordered denomination ought to have a means by which to discipline its goofier practitioners.
Holding your breath under water (Todd Pruitt) [Reformation21 Blog] 2013-10-28 10:25
Michael Jensen has written a terrific article concerning biblical sexual ethics and how the church has often become counterproductive in the ways it teaches about sex.
Jensen writes:
Or again: we speak about marriage as if it is like some kind of no holds barred festival of sex and sexual intimacy, without sexual frustration or failure, and without the discovery of mismatched desire. This puts an unreasonable level of expectation on marriages and intensifies the feeling that to be single is to be missing out. We rightly want to celebrate sex as God's good gift, to show that the Christian understanding of human sexuality is not a form of horror at the sexual. But some preachers are beginning to sound more like sex therapists than prophets. Preaching about sex is sexy. It will get you attention, if you are a preacher. But too often, by holding out the promise of marriage as a carnival of raunch it is just doing what porn does: offering a fantasy of sex instead of the real thing.
Read the entire article HERE.
Around and About (Carl Trueman) [Reformation21 Blog] 2013-10-25 09:42
Over at The Housewife Theologian, 'Nunchucks' has posted a guest column by Persis Lorenti reflecting on a recent Mortification of Spin, specifically on the question of why women in abusive relationships are often reluctant to ask for help.
The sad news from the UK is that Oliver Barclay, a very influential but understated leader in the post-war evangelical world, has died. The Independent carries an obituary of him. I never met Dr. Barclay but owe him a debt of gratitude: in the mid 1990s, he recommended my name to John Benton as someone to write occasional columns for the British evangelical newspaper, Evangelicals Now. Until that time, I had written solely for the secular academic audience, so he gave me my first break into writing of a more journalistic and non-historical vein. For that I am grateful; others are perhaps less so.
Finally, for any who have missed it, Denny Burk's excellent book, What is the Meaning of Sex, is now available.
Effective personal evangelism: understanding (Jeremy Walker) [Reformation21 Blog] 2013-10-24 07:41
To date, we have looked briefly at love, tenacity, boldness and consistency as particular features of the effective personal evangelist.
The fifth mark of the effective personal evangelist is understanding. We have said that we do not need special training - a degree course, or formal, academic, theological instruction, for example - but the effective personal evangelist does need to be man or woman of understanding. We must be men and women of God's book, praying constantly for the wisdom that only God, through his Spirit, can provide to us (Jas 1.5). We need to know the truth about ourselves, about God, and about our hearers. We must understand our own limitations, gifts, and opportunities. So we might say, and rightly, that we are not particularly well-equipped to explain the gospel to someone, but we might be particularly effective in persuading or compelling others to come and hear someone who is so able. We need to understand God himself: how and what he speaks, and how and in what ways he acts. We need to understand our hearers, which will prevent us from becoming discouraged on the one hand while also, on the other, providing us with our proper 'targets' in making Christ known. We must be properly adaptable. When Paul said, "I have become all things to all men, that I might by all means save some" (1Cor 9.22), he was not giving us a model for the church's corporate activity, suggesting that the church needs to become more like the world in order to be effective. He means that as individual believers we need to show a righteous accommodation. For example, if I am invited to go and discuss something with a Muslim in a mosque, becoming all things to all men will involve me removing any hat and shoes I am wearing. I am able to eat any food that is offered to me, without asking questions. If I am going to win someone to Christ, adapting to their expectations and circumstances where there is no principle of obedient righteousness involved and not demanding what I am not entitled to expect may give me gospel opportunities I would otherwise have lacked. Furthermore, we must unfold the truth appropriately. There is, for example, a difference - in measure - between the way that you would explain the same saving truth to a Muslim, to someone brought up in nominal Christianity, and to someone who has never heard of Jesus Christ before. You do not change the essential substance, but you might have a different point of entry, a different set of illustrations, or a different emphasis. We need discernment in these things. We need to be wise as to what we say on the first occasion when we meet someone, and how far we carry our conversation on that occasion. Some will show immediate appetite to plunge on, others will be much more wary. We need to make sure that we say what is needful, but we do not always need to say everything, and might have opportunity to return on another occasion. So, in the part of Britain where I usually work, I might be told that someone is busy and cannot talk just now. I might then suggest that another time might be more convenient. "Oh, yes, of course," is the response, often a polite British way of communicating the hope that I will never darken the door again. Consistency and tenacity will, however, return on the basis of the promise made, hoping that the same politeness will eventually provide a more convenient time. We also need to understand when the time might have come to hold our tongues and move on. We do not often, literally, put our foot in the door. We might need to wait for our opportunity. So I think of one angry atheist of distinctive appearance who - after we had first spoken to her - visited all her immediate neighbours to warn them about us. Not long after, I happened to be present when a medical crisis arose in the same spot, and obtained an opportunity to explain to those very neighbours who I was, what we were doing, and how we were operating, in a context in which they could see we had no dodgy motives. One thing that would be profitable is to make it a practice to memorise our Bibles: the grasp of the truth and the ability to handle it reactively and proactively provided by such storing up of the Scriptures cannot be underestimated. We need a working grasp of the whole Bible, a grand overview of special revelation, and we need hearts and minds well stocked with the truth. This does not mean that someone cannot be effective until they know large chunks of the Bible, but we do need accurately to know God and his truth in order to communicate the truth effectively. We would do well to read books that help us to explain the gospel, equipping us with information that we can clearly communicate, teaching us how to counter typical unbelieving responses to divine truth. We need to understand in some measure the Lord himself, his truth, our own hearts and gifts, the character and situation of the people we are dealing with, and the circumstances into which we go.
My Brief History (Iain D Campbell) [Reformation21 Blog] 2013-10-23 16:23
On my way back from a meeting in Edinburgh I picked up Stephen Hawking's newly published memoir at Glasgow airport, a short hardback aptly titled 'My Brief History', which I read on the plane journey home. Did you know he once smuggled Bibles into Russia...?
New Venue for the Philadelphia Conference on Reformed Theology (Robert Brady) [Reformation21 Blog] 2013-10-23 16:23
Keeping the Customer Satisfied (Carl Trueman) [Reformation21 Blog] 2013-10-23 08:07
The latest Mortification of Spin: Bully Pulpit is now available, dealing with the apparent inability of
the evangelical Christian world to understand how public criticism of public teaching works. If you are uncomfortable with the idea that one can criticize a Christian leader who has used their high-profile public position to say or do something that is harmful and/or wrong before one has sat down at Starbucks, bought them a latte and affirmed them in their calling, then this podcast is probably not for you.
And if you are of that persuasion but do listen to the program, please be aware that we are happy to waive our right to a latte and all that faux affirmation and instead give you permission to criticize us to your heart's content. Such is a badge of honour.
We'll keep a welcome in the hillside... (Paul Levy) [Reformation21 Blog] 2013-10-22 07:35
Effective personal evangelism: consistency (Jeremy Walker) [Reformation21 Blog] 2013-10-21 17:13
So far we have considered love, tenacity and boldness as some particular features of the effective personal evangelist.
The fourth mark of the effective personal evangelist is consistency. If tenacity is an unwillingness to let go in the face of pressure and opposition, consistency is the simple virtue of endurance over time, just keeping going, maintaining the appointed means, method, manner and matter of God's gospel ministry, going back day after day, week after week, month after month, year after year. If our tenacity can be undermined by our fear or doubt or laziness, so our consistency can be undermined by seemingly slow progress and apparent fruitlessness. But the effective evangelist keeps on going on, seeking but not demanding immediate results or definite responses the first time around. He keeps chipping away, not in the nagging sense, but there is something of the graciously dripping tap about him (giving forth living water, naturally!): he will be back running in the same places and flowing down the same channels. He will not readily give up. In Mark 4.26-29 we read that the kingdom of God is as if a man should scatter seed on the ground, and should sleep by night and rise by day, and the seed should sprout and grow, he himself does not know how. Many commentators suggest that he is not ignoring the seed, but going about his business. He takes care of the general business of the farm consistently because he knows that his task is to sow the seed and to anticipate the increase. So with the effective personal evangelist: he keeps sowing and keeps going about his business, urgent but not desperate. He keeps going on to the streets, he keeps inviting his friends to hear a man who can speak the truth, he keeps knocking on doors to tell people about the Lord Jesus, he keeps asking new neighbours into his home, he keeps engaging in the means that a lively local church uses to make Christ known, he keeps maintaining family worship so that his children might go on hearing about the Saviour. It is not always huge great hammer-blows that break the heart, but repeated taps with the gospel hammer. How long did Paul labour in Ephesus and what was his pattern of work? "For three years I did not cease to warn everyone night and day with tears" (Acts 20.31). The disciples did not fill Jerusalem with their doctrine by working in fits and starts, but by loving, tenacious, bold consistency, speaking the truth in love again and again, in this place, in the next place, to the first person and every other person, at every door and every day. You may be at times discouraged by slow progress, but you will not abandon the appointed means, method, manner and matter because there are no immediate results and definite declarations, because sometimes conversion is the result of countless hours of patient and prayerful instruction, progress comes about after weeks and months of tireless labour. We would love to see rapid results, and they are well within God's power and grace. But that is not the only way God works. Sometimes our consistency is itself a persuasive. Sometimes people will be more suddenly converted because they see and hear us going back repeatedly, or because we did not give up, and there came a day and hour when one of God's elect was in the appointed place at the appointed time, and what previously fell on deaf ears now falls into an opened ear. Whether it is in the sense of keeping doing it in the hopes of some particular barrier being broken down over time, or keeping doing it because God may change the circumstances and dynamics of a situation more suddenly, the effective personal evangelist is a consistent man. God gives us the message and we need to carry it forth, trusting in God to give the increase.
Zorro and the Musketeers (Carl Trueman) [Reformation21 Blog] 2013-10-21 16:02
I had great fun last week attending the Reformation Worship Conference at Midway Presbyterian Church near Atlanta. There was one terrifying moment when Irfon Hughes approached me and
spoke in his native language. Not having heard the Dark Tongue of Mordor spoken by a native for nearly thirty years, I had forgotten the sinister chill which always accompanies it. It was also a pleasure to be at a party on Friday night where the mysterious, tanned and masked Zorro-like swordsman, known in the P.C.A. simply as 'El Rubio,' held court.
While in Atlanta, I enjoyed staying at the house of David Hall where I was able to connect with a number of old acquaintances: Mark Ross, T. David Gordon and Terry Johnson, as well as David himself. Listening to them
talk, I was reminded of how much I owe to their writings. T. David Gordon's two works, Why Johnny Can't Preach and Why Johnny Can't Sing Hymns are brilliant examples of how the author blends theology, churchmanship and media ecology (which, quite frankly, sounds like too much fun to be a legitimate post-Fall calling) into a critique of contemporary church life. David Hall may not be as well known as T. David, but he has written numerous books. Among them, his The Practice of Confessional Subscription is outstanding and should be required reading in any class dealing with matters of church authority and confession. His syllabus for church officer training is also very good. Terry Johnson's Leading in Worship is a most useful handbook for those called to this task, one which has no obvious competitor. It is just out in a second and expanded edition and I am not able to find an online link for it as yet, but I will make one known as soon as available. In the meantime, his little book on catechizing children is now out from Banner of Truth.
All for one and one for all, as they say.
New & Notable Books [challies.com - Informing the Reforming] 2013-10-31 12:25
I am in the unique and enjoyable position of receiving copies of most of the latest and greatest Christian books and I like to provide regular roundups of some of the best and brightest of the bunch. Of all the books I have received recently, here are the ones that appear most noteworthy.
What Is the Meaning of Sex? by Denny Burk. This is one I have been looking forward to reading since I first learned about it. “Sex. We live in a world that loves it without understanding it. This book clearly explains the truth about sex and winsomely responds to society’s evolving views on human sexuality and gender. From marriage to birth control, homosexuality to singleness, What is the Meaning of Sex? sets forth a distinctly Christian perspective, equipping you to engage our confused culture with a God-glorifying vision of human sexuality.” This one comes with a long list of excellent endorsements. (Learn more or buy it at Amazon or Westminster Books)
To Live Is Christ to Die Is Gain by Matt Chandler with Jared Wilson. “Using Paul’s radical letter to the Philippians as his road map, Matt Chandler forsakes the trendy to invite readers into authentic Christian maturity. The short book of Philippians is one of the most quoted in the Bible, yet Paul wrote it not for the popular sound bites, but to paint a picture of a mature Christian faith. While many give their lives to Jesus, few then go on to live a life of truly vibrant faith. In this disruptively inspiring book, Chandler offers tangible ways to develop a faith of pursuing, chasing, knowing, and loving Jesus. Because if we clean up our lives but don’t get Jesus, we’ve lost! So let the goal be Him. To live is Christ, to die is gain—this is the message of the letter. Therefore, our lives should be lived to Him, through Him, for Him, with Him, about Him—everything should be about Jesus.” (Learn more or buy it at Amazon or Westminster Books)
Letters to Pastors’ Wives: When Seminary Ends and Ministry Begins, edited by Catherine J. Stewart. This looks like a very interesting book that will fill an important niche. “Pastors’ wives encounter special challenges as well as special joys. These letters from the seasoned wives of seasoned pastors provide empathy, wise counsel, and encouragement on a wide range of topics.” Contributors include Mary Beeke on criticism, Sarah Ascol on pastors’ kids, Margy Tripp on personal devotions and Lynn Crotts on humility. (Learn more or buy it at Amazon or Westminster Books)
Death by Living: Life Is Meant to Be Spent by N.D. Wilson. “In this astoundingly unique book, bestselling author N.D. Wilson reminds each of us that to truly live we must recognize that we are dying. Every second we create more of our past—more decisions, more breathing, more love and more loathing, all of it slides by into the gone as we race to grab at more moments, at more memories made and already fading. We are all authors, creators of our own pasts, of the books that will be our lives. We stare at the future or obsess about the present, but only the past has been set in stone, and we are the ones setting it. When we race across the wet concrete of time without purpose, without goals, without laughter and love and sacrifice, then we fail in our mortal moment. We race toward our inevitable ends without artistry and without beauty.” (Learn more or buy it at Amazon or Westminster Books)
Preparing for Marriage God’s Way: A Step-by-Step Guide for Marriage Success Before and After the Wedding (2nd edition) by Wayne Mack. “Preparing for Marriage God’s Way is a marriage counseling resource that uses thoughtful self-examination to reveal the personalities, background, and expectations that you and your partner are bringing to your union. Through rigorous Bible study, you will learn about God’s expectations for marriage and be equipped with his solutions for dealing with typical marriage conflicts. Three follow-up lessons after your marriage help you to reflect on all that’s happened after you said, ‘I do’.” (Learn more or buy it at Amazon)
Dictionary of Jesus and the Gospels (2nd edition) edited by Joel B. Green, Jeannine K. Brown & Nicholas Perrin. This looks like a very helpful reference work. “The second edition of the Dictionary of Jesus and the Gospels is a thoroughly reconstructed and revised version of the critically acclaimed 1992 first edition. Since that groundbreaking volume was published, a wave of Jesus and Gospel scholarship has crested and broken on the shores of a new century. Jesus has been proposed as sage, shaman, revolutionary, marginal Jew, Mediterranean peasant or a prophet of Israel’s restoration. The non-canonical Gospels have been touted, examined and reassessed. There are revised understandings of historiography, orality, form criticism, empire and more. The second edition of the DJG amply weighs and assess the gains and shortcomings of this new scholarship.” (Learn more or buy it at Amazon)
And how about you? Are there some new and notable books that you’ve added to your reading list? Is there anything I’m missing?

Be a Zealous Christian! [challies.com - Informing the Reforming] 2013-10-31 07:58
Over the past few weeks Dr. Joel Beeke and I have been teaming up to work our way through a portion of his massive new work A Puritan Theology. We have not been reading the whole book, but just the final eight chapters which deal with practical theology, the “so what?” of systematic theology.
This week we read chapter 58 which discusses the Puritans and zeal. I asked Dr. Beeke a few questions related to the Puritans and this word that seems to have fallen into disuse today.
TC: When the Puritans spoke of zeal, what were they referring to?
JB: By zeal they meant the fruit of the Spirit, especially love, exercised to a high level in the soul and activity of life. Thomas Manton said that godly zeal is “a higher degree of love,” indeed the burning of divine love. Manton wrote, “Zeal will readily set us a-work to do all we do willingly, freely, and cheerfully” (2 Cor. 9:2). It is distinguished from “carnal zeal” by its lack of hatred and bitter envy (James 3:14), its direction by a true knowledge of God’s Word (Rom. 10:2), and its keeping its focus on piety of the heart instead of superstitious externals (Matt. 23:23; Rom. 14:17). Yet zealous love does include a holy “indignation” because when we love something strongly then we hate all that is against it. The strength of zealous love moves Christians to deny themselves and press on despite resistance. It fills them with “holy grief and anger” whenever God’s truth, God’s worship, or God’s servants are violated.” For example, David wrote, “My zeal hath consumed me, because mine enemies have forgotten thy words. Thy word is very pure: therefore thy servant loveth it. I am small and despised: yet do not I forget thy precepts” (Ps. 119:139–141).
TC: Zeal seems to have been an important concept and an important component of Christian character to the Puritans. What has happened to zeal? Have we simply replaced the word with another, or have we lost the whole concept and emphasis?
JB: Zeal can never completely disappear from true Christianity, for it is, as Manton said, “a fruit of Christ’s death” (Titus 2:14), partly because the marvelous display of Christ’s love inflames His people to love Him, and partly because Christ purchased the gift of the Spirit to make us zealous to serve Him (Titus 3:5–6).
People may use different words for zeal. I hear some Evangelicals use the word passion in a way similar to how the older writers spoke of zeal. The Bible does not use this word in this manner (“passion” in Scripture refers to either suffering or out-of-control desires), but it seems to me that they aim to communicate a similar idea. The older generation would talk of being on fire for the Lord, which is really the meaning of the biblical word “fervent” (Acts 18:25; Rom. 12:11). So the concept is still there.
The danger we face today is that the courage, strength, activism, and resolve of zeal offend our culture of feminized men and tyrannical tolerance.
TC: If the Puritans were to take a pulse on Reformed Evangelicalism today, how might they challenge us in regards to zeal?
JB: The Puritans might challenge us to consider more soberly the danger of false zeal. In an age when the love of so many has grown cold, we may get excited whenever we encounter someone who is “fired up” for God. Stephen Charnock said, “Nothing is so great an enemy to true Christianity as ignorant zeal.” Zeal must come with the wisdom from above which is pure, humble, and compassionate, or it will end in bitterness, disorder, and sin (James 3:13–18). Zeal is only as good as the doctrine and godliness which inspires it.
The Puritans might also call us to implement a more extensive zeal that covers all of life. We may be tempted to confuse zeal with excitement in meetings, especially large meetings with music and emotions raised to fever pitch. Christians tend to associate zeal with specific activities such as singing and evangelism. The Puritans emphasized the Reformation doctrine of vocations: everyone serves God in his or her own calling at work, school, or in the home. John Preston wrote, “If you will show that you love the Lord Jesus, do the works that belong to your particular place; for every calling hath a particular work in it: if you love the Lord, be diligent in that way, in that calling which Christ hath given you do to him service in: and herein you shall show your love.”
The Puritans would perhaps remind us that zeal is not about excitement so much as it is about perseverance in doing good out of a steady love for God. Manton said, “To be zealous of good works is to be constant to the end… . Zeal is not like fire in straw.”
TC: How can we increase the zeal in our lives and in our churches?
JB: First, look to Christ to increase your faith and love. Christ alone can give us life, and cause us to grow. Preston said, “We know nothing but what we are taught by him as a Prophet; whatsoever we do is lost labour, except it be made acceptable through him as a Priest; we are able to overcome no lust, to do no duty but through the power we have from him as a King.”
Second, use the means of grace diligently, especially prayer, the Word, and the sacraments. Christ is the fountain and the means are the “conduit-pipes,” as Preston said, and therefore we must not break the pipes off the fountain, nor expect to receive grace except by drinking at the pipes.
Third, examine yourself to see if your heart is divided. No woman will grow in her love for her present husband if her heart clings to her former husband. Preston wrote, “We must labour to be divorced from all other husbands … weaned from all earthly things, to which [our hearts] are too much wedded.”
Fourth, exercise whatever zeal you have by obeying God’s Word with all boldness. As Preston said, we must not let the grace God has given us sleep within, “but we must draw it forth to action, bring it out to practice, and that upon all occasions.” This is especially the case when obedience involves some “hazard” of potential “losses and crosses.”
Fifth, do not be ashamed when people think you are strange and deluded. Manton reminds us that people who are serious about godliness, deny themselves for Christ, and are zealous for a good cause are commonly counted as afflicted with “folly and madness” (Jer. 29:26–27; Acts 26:24; 2 Cor. 5:13). They said Christ was strange and deluded too (Mark 3:21; John 10:20)!
TC: Would the Puritans say it is a pastor’s responsibility to fan the flames of zeal in his church? How would he do this?
JB: The pastor must do what he can, as God has commanded him. He is instrumental in bringing the public means of grace to the people. He also must seek to be zealous in his Christian life (and not just his ministry). Pastors may find read more about this in the book I co-authored with Terry Slachter, Encouragement for Today’s Pastors: Help from the Puritans.
At the end of the day however, each of us is responsible before God for his own life. Never blame your pastor for your lack of zeal. William Fenner said, “The minister may be lively, and yet the people dead. The Lord tells us that Ezekiel had a stiff-hearted people (Ezek. 2:4), yet he was not to be blamed, themselves were in all the fault.”
If you are reading along with us, be sure to read Chapter 59 (“Practical Lessons from Puritan Theology Today”) by next Thursday. Then simply check in here to see what Dr. Beeke has to say about it.
The purpose of this project is to read classics together. Please feel free to leave a comment below or to provide a link to your own blog if you have discussed this week’s chapter there.

A La Carte (10/31) [challies.com - Informing the Reforming] 2013-10-31 06:33
Here are a few new Kindle deals: A Walk with God by R.C. Sproul ($3.99); Crossway has a few volumes of the excellent Preaching the Word commentary set on sale for $3.99 each: Deuteronomy, 1-3 John, Proverbs, and The Song of Solomon. Don’t forget about the huge lists of deals from Tuesday and Wednesday.
Will We Drink Coffee? - As Randy Alcorn says in this article, a question like “Will we drink coffee in heaven?” is “a revealing test of whether we’re more influenced by biblical teaching or Christoplatonism.”
Luther and the Reformation - In honor of Reformation Day, here’s a free teaching series from Ligonier. And while on the subect of Reformation Day: What Is Reformation Day All About?.
Thy Word Is Still Truth - Thy Word Is Still Truth is an important new volume on Scripture and, for a limited time, Westminster Books has it at a deep discount.
The Gift of Fasting - “Fasting isn’t usually a word that conjours up ideas of blessing. In western cultures, it’s more associated with hair shirts, self-flagellation, and shaved heads. In the Protestant church, we look at it more as a tool to refocus. But it’s a whole lot more than that.”
The Call to Ministry - Bobby Jamieson has begun an interesting-looking series on the call to ministry. “This series of posts is geared toward men considering or pursuing pastoral ministry. It’s also geared toward the churches which encourage and assess them—which, in one way or another, is every church.”
A Call to Resurgence - Andrew Wilson, writing for The Gospel Coalition, reviews Mark Driscoll’s A Call to Resurgence. He says it is “sometimes insightful, sometimes amusing, sometimes stirring, and sometimes exasperating.”
Halloween: Trick or Treat? - You may enjoy this excellent video from 10ofThose.
The real horror of being outside of Christ is that there is no shelter from the wrath of God. —Eric Alexander


One People, One Voice [challies.com - Informing the Reforming] 2013-10-30 08:26
With the entrance of sin into the world and into our hearts, we have gained some remarkable (and remarkably sad) abilities. One of those newfound abilities is the capacity to lose our wonder, to grow cold to even the most beautiful things. Things that once inspired us, that once moved us, that caused us to marvel or to cry out in praise—even these things can grow stale over time; even these things can become old. One of the great joys and great promises of eternity is that in heaven we will never lose our wonder but will, to the contrary, enjoy ever-increasing wonder and ever-increasing joy. What moves us today will move us even more tomorrow and what causes us to marvel tomorrow will bring even greater wonder in the future. C.S. Lewis captures just a little bit of this in the closing sentences of his Narnia chronicles:
And for us this the end of all the stories, and we can most truly say that they all lived happily ever after. But for them it was only the beginning of the real story. All their life in this world and all their adventures in Narnia had only been the cover and the title page: now at last they were beginning Chapter One of the Great Story which no one on earth has read: which goes on for ever: in which every chapter is better than the one before.
“Every chapter is better than the one before.” If only that were true here and now. It should be, but it is not.
Just recently I realized that I had somehow lost much of the joy in one of life’s great pleasures.
I have had the experience a few times. I have walked into a building—a church or a school or a community center where a group of Christians meets—and I have heard distant singing. I’ve gone to investigate, walking quietly toward the sound, trying to track it down. And as I wandered the halls, I eventually found a gathering of Christians, expressing praise to God through song.
What continues to fascinate me is that as I got closer to the source of the singing, I began to hear distinct voices. When I in the middle of that group, I could begin to make out this person’s tenor and that person’s alto. I could hear that this man was singing melody and that woman was singing the matching harmony. That song was made up of many voices and many parts, each person contributing his or her part to the whole.
But then I would step back again, I would walk a little bit further away, and as I heard that praise from afar, it was as if one voice was crying out in praise to God.
And it was this corporate dimension to singing, this one voice, that I neglected. So much of the beauty of singing praises to God is experiencing a hundred people, or a thousand people, praising God all together in one voice. There is much we do as individuals in the corporate worship services, but there is also this dimension of gathering as a group, as a body, as a single spiritual organism. Singing is something we do together. We sing to encourage one another, we sing to teach and admonish one another, but ultimately, we sing so we can praise God together, as God’s people. Many individuals, but one people with one voice. It’s a beautiful, wondrous thing.

A La Carte (10/30) [challies.com - Informing the Reforming] 2013-10-30 06:14
Here are a few more Kindle deals (see yesterday’s huge list here): Pilgrim Theology by Michael Horton ($7.99); The Christian Faith by Michael Horton ($19.99); Systematic Theology by Wayne Grudem ($19.99); The Gagging of God by D.A. Carson ($6.99); Center Church by Tim Keller ($8.99); Church History (Volume 2) by John Woodbridge ($9.99); A Better Way by Michael Horton ($3.99); 10 Who Changed the World by Daniel Akin ($2.99); Breaking the Islam Code by J.D. Greear ($1.99); Reading the Gospels Wisely by Jonathan Pennington ($4.99); What Christians Believe by Jonathan Gould ($3.99); What the Bible Means to Me by Catherine MacKenzie ($2.99).
Parents, Require Obedience - John Piper: “I am writing this to plead with Christian parents to require obedience of their children. I am moved to write this by watching young children pay no attention to their parents’ requests, with no consequences.”
Quotes on Creeds and Confessions - Here are ten good quotes from Carl Trueman on the importance of creeds and confessions.
PrayerMate for Android - PrayerMate is a fantastic prayer app. Currently it’s only available for iOS, so if you’re an Android user, you may want to look at this Kickstarter project.
The Myth of “I’m Bad at Math” - No, you’re not. You just didn’t try hard enough.
5 Differences Between Catholic Theology and the Gospel - “With Reformation Day this week, it is a good time to remind ourselves of what exactly the differences are between the Roman Catholic Church and Protestants. Certainly on just about every single area of theology there are differences, but here are what I think are the five most glaring and significant issues that separate the Catholic Church from the gospel of grace…”
Remove an Emphasis - I continue to be a big fan of Bill Mounce’s series on Bible translation conundrums. Here he expresses concern over a missing emphasis.
There are some of your graces which would never be discovered if it were not for your trials. —C.H. Spurgeon


Let's Ask John MacArthur Some Hard Questions [challies.com - Informing the Reforming] 2013-10-29 13:03
John MacArthur’s Strange Fire conference has come and gone and the book will be shipping in just a couple of weeks. Whatever you felt about the conference, there is little doubt that a lot of work and a lot of discussion remain as we, the church, consider the miraculous gifts of the Holy Spirit. In the aftermath of the event, and with the book on its way, I know a lot of you have questions you would like to ask Dr. MacArthur. These may be tough questions. They may be critical questions.
I have asked, and he is eager to answer your questions.
So if there is something you would like to ask John MacArthur about the Strange Fire conference or book, if you have a question, and perhaps especially if you have a tough question, please leave a comment below. I will collect some of them and send them through so he can provide an answer (which will appear here in a week or two).
I would ask only that you pause for at least one moment before you hit the “Post” button. In that moment, consider your tone and perhaps whisper a quick prayer for wisdom. In that regard it might be helpful to imagine that Dr. MacArthur is sitting across a table from you, willing to hear your concern. And then ask that question, even if it is a tough one.
Note: Below each comment you will see “vote up” and “vote down” buttons represented by an up arrow and down arrow. Feel free to make use of those buttons to help elevate another person’s question (though there’s no guarantee I will send through those questions…).

18 Things I Will Not Regret Doing With My Wife [challies.com - Informing the Reforming] 2013-10-29 07:59
Last week I shared 18 Things I Will Not Regret Doing With My Kids, and the time I spent writing that article got me thinking about the fifteen years I’ve been married to Aileen (and the three years before that when we dated). I felt it was only right to think of another eighteen things, and this time to do so in her honor.
Here are 18 things I know I will never regret doing with my wife.
1. Praying with her. It took too long for the two of us to begin to really pray together; even now, we have a long way to go. But we have learned the importance of praying together and never regret the times we spend together before the Lord.
2. Dating her. We have all heard a thousand times how important it is to keep dating, even after getting married. This is easier said than done when the children are young and high-maintenance, but we have found it much easier now that the kids are just a little bit older. I have never regretted these times alone together.
3. Serving with her. While the majority of my relationship with Aileen is lived out face-to-face, we have always worked very well together side-by-side. We’ve planned and executed all kinds of events and programs in the past, and inevitably grow closer as we have done this. I never regret the time we spend serving together.
4. Looking back with her. Some of our sweetest times are spent looking at relics of days gone by—the silly journals we kept when dating, the photos of our wedding, the children when they were infants. Looking back is a genuine pleasure and we never regret that time together, remembering what the Lord has done and where he has brought us.
5. Leading her in love. I am convinced God has called me to lovingly lead my wife. This kind of leadership does not come easy to me, but I know there is a high cost to refusing to take it up. I never regret leading Aileen, when I lead with her good as my goal and with Christ as my model.
6. Buying her flowers. I am fifteen years into marriage and still feel sheepish carrying a bouquet of flowers through a parking lot. But the flowers continue to be special, she continues to love them, I continue to enjoy giving the gift. I will never regret showing love that way.
7. Asking her forgiveness. It is a strange and ugly reality that the person I love most is the person I sin against most often. I have never-ending opportunities to ask her forgiveness. While it requires choking down my pride, I know I will never regret asking her to forgive me when I have sinned against her.
8. Forgiving her. Of course it works both ways, and she sins against me as well. Like me, she can struggle with asking for forgiveness, so when she does ask, I never regret immediately and sincerely forgiving her, and putting that offense out of my mind.
9. Holding her hand. It is so easy to allow what used to be special to become unremarkable and forgotten. Holding hands is one of those sweet habits that can so quickly be lost. I will never regret reaching out and walking with her, hand-in-hand.
10. Planning her hobby time. Aileen gives so much of herself to home and family, but tends to be at her best when she has a hobby to give some of her time and attention to. I never regret the time we carve out to plan how she can give time to the hobbies she loves.
11. Washing her with the Word. The book of Ephesians makes it clear that one of a husband’s joyful responsibilities is washing his wife in the water of God’s Word. As our marriage has progressed we have seen more and more clearly the value and beauty of doing this very thing. I will never regret those times we spend together, hearing from God through his Word.
12. Listening to her. I am far too quick to give my own opinion, to make excuses, to speak without really listening and hearing. But I am learning that I will never regret the times when I patiently listen and allow Aileen to speak without interruption, without interjection, without having me become all defensive.
13. Reading with her. If you want to talk about compatibility within marriage, well, Aileen and I are very incompatible when it comes to the books we love to read. But when we do find one of those books and when we commit to reading it together, I never regret the time or the effort.
14. Delighting in her. In all the sin, stress and strain life can bring, it is so easy to lose that sense of wonder and delight in the gift of a wife. I will never regret thinking about her, thanking God for her, and increasing my delight in her.
15.Enjoying shared interests. One of the first things I did when when I began dating Aileen was learn to like tennis; that was just the first of many interests we learned to enjoy together. I have never regretted learning to enjoy something she loves for her sake and for the sake of our relationship.
16. Worshipping with her. One of my great joys in life is worshipping the Lord side-by-side with the one person I love more than any other. This is a little foretaste of heaven, just a glimpse of eternity, where we will worship him perfectly forever. I never regret prioritizing church and worshipping with Aileen.
17. Getting away with her. We love our family vacations with the five of us sprawled out at the beach or huddled in a cabin. But Aileen and I also find great benefit in vacations alone, whether that is a couple of days somewhere nearby, or a week somewhere far from home. I will never regret interrupting normal life for these sweet times together.
18. Saying I love you. Yes, even the “I love you” can become an empty habit rather than a meaningful declaration. When I pause for just a moment, when I think about what I am saying, that little phrase takes on much greater depth of meaning. I never have and I never will regret looking Aileen in the eyes and saying, “I love you.”
The joy of this list is that I could so easily have come up with another eighteen items, and another eighteen beyond that. The Lord has blessed me so far beyond what I deserve.

A La Carte (10/29) [challies.com - Informing the Reforming] 2013-10-29 06:14
There are a lot of new Kindle deals today: The Next Story by me ($3.99); Historical Theology by Gregg Allison is a great deal at $5.99; Christian Beliefs by Wayne Grudem ($3.99); Worship by the Book by D.A. Carson ($2.99); Don’t Stop Believing by Michael Wittmer ($2.99); Gospel Coach by Scott Thomas & Tom Wood ($2.99); A God-Sized Vision by Collin Hansen & John Woodbridge ($3.99); Telling the Truth edited by D.A. Carson ($2.99); Is Hell for Real or Does Everyone Go to Heaven? edited by Christopher W. Morgan & Robert A. Peterson ($2.99); Culture Making by Andy Crouch ($4.99); A Place for Weakness by Michael Horton ($2.99); For Calvinism by Michael Horton ($2.99); Unleashing the Word by Max McLean ($2.99); Preaching & Preachers by Martyn Lloyd-Jones ($2.99); Bound Together by Chris Brauns ($4.99); Joni & Ken by Joni & Ken Tada ($2.99) How to Read the Bible Through the Jesus Lens by Michael James Williams ($2.99); The Hardest Sermons You’ll Ever Have to Preach by Bryan Chapell.
A More Boring Writer - Matthew Lee Anderson offers nine tips on how to become a more boring writer.
Strange Fire - New Testament scholar Tom Schreiner reviews MacArthur’s Strange Fire (book). “I have traveled a road in which I was a cessationist in my early years as believer. I then became a non-cessationist and taught accordingly for a number of years. Finally, in the mid 1990s I slowly returned to a cessationist position. I am sympathetic, then, to the case MacArthur makes for cessationism.” He offers both commendations and critique.
Perils Facing the Evangelical Church - R.C. Sproul offers his take on the three most critical perils the church faces today.
Family: The Original Small Group - Timothy Paul Jones: “In my research for the book Family Ministry Field Guide, I found that only one-third of churched parents read or discussed Scripture with their children at least once a week. If that’s the predominant pattern, it seems unlikely that the typical teenager in an evangelical church would identify his or her parents first as teachers of God’s Word.”
A Plea to Pastors and Elders - Aimee Byrd: “Pastors, elders, is there something more we could be doing in the church to equip the congregation with discerning reading skills? Good, Christian people are being deceived. Sadly, sometimes it is those in leadership who recommend such books. The so-called Christian author can actually be a potential predator of orthodoxy in the church.”
Christ-Centered Preaching and Teaching - Here is a new, free e-book from The Gospel Project.
The great of this world are those who simply loved God more than others did. —A.W. Tozer


Best Commentaries on Proverbs [challies.com - Informing the Reforming] 2013-10-28 12:59
Series Introduction: I live in a small house. I work in a small office in a small church. For those reasons and others I will never have a huge library. When I add a book I almost always remove a book, a practice that allows me to focus on quality over quantity. Over the past couple of years I have focused on building a collection of commentaries that will include only the best volumes on each book of the Bible. I know when I’m in way over my head, so before I began I collected every good resource I could find that rated and reviewed commentaries. I studied them and then began my collection on the basis of what the experts told me. Since I did all of that work, and since I continue to keep up with the project, I thought it might be helpful to share the recommendations.
My focus is on newer commentaries (at least in part because most of the classics are now freely or cheaply available) and I am offering approximately 5 recommendations for each book of the Bible, alternating between the Old Testament and the New. Today I have turned to the experts to find what they say about Proverbs.
Bruce K. Waltke - The Book of Proverbs: Chapters 1-15; The Book of Proverbs: Chapters 15-31 (New International Commentary on the Old Testament). The clear consensus among the experts is that Waltke’s massive two-volume set is the absolute best commentary on Proverbs. Derek Thomas refers to it as “a definitive work” and “a lifetime achievement.” It is the result of two decades of thoughtful reflection and the introduction alone is worth the cost of the set. It’s the place to begin for Proverbs and a must-have for any pastor. (Amazon: Volume 1, Volume 2; Westminster Books: Volume 1, Volume 2)
David A. Hubbard - Proverbs (The Preacher’s Commentary). To some degree, all other commentaries are considered supplementary to Walke’s set, so when it comes to Hubbard, Keith Mathison says, “Those who are preaching or teaching through Proverbs will want to supplement Waltke’s work with a commentary that reflects on various issues related to application. Hubbard’s work is the best place to turn for this. While not as comprehensive as Waltke, Hubbard does not neglect addressing the difficult questions, all the while remaining clear and readable.” (Amazon)
Tremper Longman III - Proverbs (Baker Commentary on the Old Testament Wisdom and Psalms). Longman is a notable Old Testament scholar who also pens a helpful Old Testament Commentary Survey that I’ve relied on for this “Best Commentaries” series. His volume on Proverbs is said to be detailed but still readable, scholarly and yet still accessible. At 608 pages, it is quite thorough and its main focus is on the theological and ethical message of the book. (Amazon, Westminster Books)
Derek Kidner - Proverbs (Tyndale Old Testament Commentaries). All of the commentators on the commentaries regard Kidner’s as an excellent commentary, though one limited by the constraints of the TOTC series which requires short, popular-level volumes. Longman says, “This small commentary is packed with helpful insight and comments on the text. It is exegetically sensitive, theologically helpful, and orthodox.” This is a great option for devotional reading, for simple Bible studies, and for providing application. (Amazon, Westminster Books)
Raymond C. Van Leeuwen - “Proverbs” in The New Interpreter’s Bible. There may be no need for a fifth volume on Proverbs if you’ve got each of the ones above, but if you need one more, consider Van Leeuwen. Mathison says, “Van Leeuwen is a recognized scholar in the field of biblical wisdom literature, and his understanding of this genre is reflected in his commentary. There is more reflection on the theology of the book here than in most commentaries.” You will probably have some difficulty tracking it down and may need to visit a theological library. (Amazon)
And let me close with a question: What are your preferred commentaries on Proverbs? Are there some you’ve found particularly helpful?

The Safe Place for Our Kids' Shameful Questions [challies.com - Informing the Reforming] 2013-10-28 08:30
Apart from getting my teeth bashed in by a hockey stick and that wet-to-the-skin oh-why-did-I-wear-jeans hiking trip through Gatineau Park, it is one of my few vivid memories from eighth grade: Jeremy was, like me, a new student at the school that year. Jeremy, like all boys our age, was curious about female anatomy. So Jeremy did the one thing he knew he could get away with: Giggling all the while, he went to where the Webster’s Dictionary was stationed at the back of the room, looked up the word vagina, and finally solved one of those mysteries that had been perplexing him. In retrospect, I guess he didn’t really solve it, because the dictionary definition was undoubtedly rather drab and technical for a thirteen-year-old mind. It was correct and official but probably rather unsatisfying. Still, it sated him for a time.
Maybe his parents had never had the talk with him. That talk with him. Jeremy’s parents were not alone in skipping it. Some parents could never bear to do it. Some relied on guidance counselors or middle school health teachers. Some, red-faced, handed their kids a book and said, “If you’ve got any questions, just ask.” Jeremy’s natural curiosity led him to do a little research and the resource readily available to him was the dictionary. No harm done. His innocent question was met with an innocent answer.
Times have changed, and yet at the same time, very little has changed. Our children are still curious. Our children still have questions they are uncomfortable asking their parents. But today our children have a whole new way of finding answers.
Today we train our children from a young age that Google is the place to go for answers. Whose is the fourth face on Mount Rushmore? Google it! What kind of reviews is that new movie getting? Google it! When is the next iPod going to be released? Google it! Should I be concerned about that mole on my back? Google it!
One of the facts I’ve learned since writing a book on pornography, and since having the opportunity to write articles for various sites and publications, is that children today are taking their innocent questions to Google and, all too often, receiving decidedly non-innocent answers.
Young girls are testifying that they just wanted to know a little bit about human anatomy—the curious questions of young teen or even pre-teen girls—and were met by the most vulgar pornography or by invitations to begin exploring their own bodies. Where a dictionary definition was the best you could get in my generation, the digital generation is finding graphic, pornographic, high-definition video. These searches had led to sexual awakenings and sexual addictions which in turn led to deep shame and stunted spiritual growth. And all the while these girls were simply doing what had been modeled to them from a young age: taking their questions to Google.
Young men are saying much the same, that few of them set out to find pornography; it just kind of happened as they followed the path of their natural, hormone-driven curiosity. Innocent questions were answered by hardened hearts and seared consciences. And if an innocent question can so quickly and easily lead to such perversion, how much more the inevitable non-innocent questions a teenaged boy’s brain will also dream up?
Parents, the simple fact is that our children take their shameful or embarrassed questions to a safe place. And today the ultimately safe places are Google or elsewhere on the Internet. We have tacitly trained them to do this, we have encouraged them to do this, and yet we are strangely oblivious to it when the nature of those questions is anatomical or sexual.
The pornographers are not oblivious. Not at all. They have learned that young people will ask these questions and they respond by building and positioning their pages in such a way as to be the first to answer them. Like McDonald’s and Pepsi, they know that if they can get the kids when they are young, they will have customers for life. Brand loyalty and all of that. So they are only too happy to tell your children what they want to know, and a whole lot more besides.
This lays down a challenge for parents. More than ever, you need to open the channels of communication with your children so they know you are safer and wiser even than the search engines. More than ever, you need to ask them questions and to invite them to ask you questions. And all the while, you simply have to be aware of what they are searching for, and what questions they are taking to the all-knowing Google.

A La Carte (10/28) [challies.com - Informing the Reforming] 2013-10-28 05:56
Here’s a handful of Kindle deals to start your week: The Man of Sin by Kim Riddelbarger ($3.99); Heroes and Monsters by Josh James Riebock ($1.99); Magnifying God in Christ by Tom Schreiner has fallen a bit further to $4.39. Playing God by Andy Crouch is brand new; I haven’t read it yet, though I hope to ($4.99).
Dude! - I may have a slightly abnormal interest in etymology, so maybe I’m the only one who cares about this. But here’s an article on the origins of the word “dude.”
Is Chastity Even Possible? - “Is the teaching of the Bible on human sexuality even possible to fulfil? Is it simply inhuman to say that the only context for human sexual expression is a lifelong marriage between a man and a woman?” Michael Jensen answers.
The Verbal Backspace Button - We’ve all wished we could hit “backspace” when we’ve uttered a careless word. Hence this article from Lindsey.
Amish Farm - It is rare that a photographer is allowed to take pictures of the Amish. This one was allowed and has quite an interesting photo gallery as a result.
Strange Fire - All of the audio for the Strange Fire conference is now available if you want to catch up.
Airline Seats - I knew this was happening, but it’s good to see the proof! “Airlines’ push to lure high-paying fliers with flatbed business seats and premium economy loungers is leaving economy-class passengers with less space.”
We discern the growth of grace as the growth of plants, which we perceive rather to have grown than to grow. —John Flavel


The Philanthropists: William Colgate [challies.com - Informing the Reforming] 2013-10-27 06:22
Our next Christian philanthropist, William Colgate, founded a company that has placed a well-known product in many of our homes even today—Colgate toothpaste. Born in Kent, England in 1783 to Robert and Sarah, Colgate migrated with his family to Maryland in 1798 because of his father’s political sympathy for the American War of Independence and the French Revolution.
While in Maryland, Colgate helped his father manufacture soap and candles, but it was after he moved to New York City in 1804 that he became an apprentice to a soap-maker and in this position learned the manufacturing business. In 1806 he founded his own starch, soap and candle business on Dutch Street in New York City, and this small shop eventually grew into a massive and thriving corporation. A skilled and principled businessman, Colgate would in due time become one of the wealthiest and most generous men in New York City.
In his early days Colgate attended a Presbyterian church pastored by Rev. Dr. Mason, one of the most prominent preachers in New York. In conjunction with his business success, Colgate was highly esteemed amongst the church members and he played an active part in the church. However, after corresponding with his Baptist father, Colgate came to reject infant baptism and, having done so, joined First Baptist Church of New York where Rev. William Parkinson baptized him in 1808. He remained an active, generous churchman throughout his life. He served as a deacon (he was affectionately known as Deacon Colgate) and became known not only for financial generosity, but also for faithfully serving the people of his church.
In 1811, Colgate married Mary Gilbert and together they had three sons, Robert, James and Samuel. Colgate eventually served on the Board of Managers of the American Bible Society (ABS) as its treasurer. Later, after dealing with controversial matters within the ABS, Colgate helped organize the American Bible Union in 1850, and again he served as the treasurer of that society until his death.
Beside serving the Bible societies, Colgate also supported Hamilton Literary and Theological Institution (later Madison University and Theological Seminary). Likewise, he regularly gave to the Baptist Missionary Union, and he even fully funded a foreign missionary on his own. Colgate made financial provisions for his aging parents by purchasing a farmhouse in a neighboring county, and he supported them financially the rest of their lives. Because of his kind and generous personality (along with Mary’s congenial spirit), his home was known as an especially pleasant and welcoming place to be. Throughout his life, Colgate attributed his success in business and ministry to the principles and truths taught in the Bible.
In 1857, Colgate died at the age of 74 and his son Samuel succeeded him as President of Colgate & Company and for a time it remained a family business; in 1928 the company was sold to Palmolive-Peet to become the Colgate-Palmolive-Peet Company (though Peet was subsequently dropped). As long as the family was involved in the business, they maintained the founder’s spirit of generosity. Samuel and James were both benefactors of Madison University and Theological Seminary. Because of the family’s great financial support, the school was renamed Colgate University in 1890, seven decades after the Colgate family initially got involved with the institution.

Weekend A La Carte (10/26) [challies.com - Informing the Reforming] 2013-10-26 06:22
There are a couple of new Kindle deals to be aware of today: Contend by Aaron Armstrong ($0.99); 10 People Every Christian Should Know by Warren Wiersbe (free).
Initiating and Declining - This is a very good article from Brad Hambrick. He talks about initiating and declining sex within marriage and offers some sound, practical counsel.
Better Selfies - Here are 4 quick tips for taking better selfies.
9 Things About Down Syndrome - Joe Carter: “October is Down Syndrome Awareness Month. Here are nine things you should know about the condition.”
Greatest Innovations Since the Wheel - Lists like this are always a little bit silly, but interesting nonetheless.
The Elusive President - I enjoyed this article at the New York Times about JFK and the surprising lack of good biographies of the man. “An estimated 40,000 books about him have been published since his death, and this anniversary year has loosed another vast outpouring. Yet to explore the enormous literature is to be struck not by what’s there but by what’s missing. Readers can choose from many books but surprisingly few good ones, and not one really outstanding one.”
Redeeming Social Media - I enjoyed this interview with Nathan Bingham as he discusses Christians, ministry, and social media.
We must come to good works by faith, and not to faith by good works. —William Gurnall


Peace of Conscience Before God [challies.com - Informing the Reforming] 2013-10-25 07:39
Over the past few weeks Dr. Joel Beeke and I have been teaming up to work our way through a portion of his massive new work A Puritan Theology. We have not been reading the whole book, but just the final eight chapters which deal with practical theology, the “so what?” of systematic theology.
This week we read chapter 57 which discusses the Puritans and casuistry. I asked Dr. Beeke a few questions related to the Puritans and this strange word that I had never encountered before.
TC: I guess we need to begin here: What is casuistry and why did the Puritans focus on it?
JB: Casuistry is teaching people how to know what God wants them to do in specific situations, and how to live with peace of conscience before God. It addresses particular “cases of conscience” or ethical and spiritual questions. The Reformation of the sixteenth century brought a renewed understanding of justification by faith alone and sanctification by the Holy Spirit, but these very doctrines raised questions such as, “How do I know if I have justifying faith?” or, “What does it mean to please God at my job?” Therefore the Puritans, as heirs of the Reformation, developed answers to such questions based upon the Word of God.
TC: What was the place of counseling for the Puritans? Was it something they did primarily in the corporate worship service or was it done one-on-one and in private?
JB: The answer is both. William Perkins, who wrote a foundational treatise on preaching, said that the preacher must apply the law and the gospel to the several specific spiritual conditions in which people find themselves. Someone who is ignorant and unteachable needs far different treatment than someone broken under the guilt of sin. Some listeners need milk, and others strong meat. Fifty years later the Westminster Assembly, in the Directory for the Publick Worship of God, said the minister “is not to rest in general doctrine,” but “to bring it home” in specific applications, including teaching the truth, refuting errors, exhorting for obedience, warning against sin, applying comfort, and directing self-examination. As a result of such an approach to preaching, Puritan sermons were full of practical counseling.
At the same time, the Puritans recognized that a pastor must counsel families and individuals in a more personal way. Some Puritans did more of this than others. John Owen said some people in a church will face particular spiritual difficulties, such as “the terror of the Lord” on those convicted but not yet converted, backsliding into sin after conversion, great and long-term afflictions, feeling abandoned by God, and horrible temptations from Satan. It is part of a pastor’s calling to understand their cases and the right spiritual medicines to heal them, to give such people attention and concern with patience and tenderness. Personal work is very fruitful both for comfort and rebuke. Richard Baxter said, “I have found by experience, that an ignorant sot that hath been an unprofitable hearer so long, hath got more knowledge and remorse of conscience in half an hour’s close discourse, than they did from ten year’s public preaching.” Preaching the Word is the primary means of grace, but personal counseling plays a significant role as well.
TC: Casuistry was a major emphasis for the Puritans, but it is not just the word that has faded—it is also the practice. What have we lost? What would we gain if we recovered it?
JB: We have nearly severed experience and practice from biblical doctrine. On the one hand, this has sometimes resulted in preaching that rests content in mere teaching or only the most general of applications. Congregations become teaching centers, producing Christians who are informed but do not know how to relate their experiences and particular battles for sanctification to the Word. A return to Puritan casuistry would make preaching much more practical. It would also display more of God’s wisdom, as people feel that “Aha! That’s me!” because their hearts resonate with how the Bible describes various spiritual experiences.
On the other hand, this divorce between the Christian heart and the Christian head has sometimes resulted in counseling that is not grounded in the sound doctrines of Scripture but instead follows secular psychology (with a few verses thrown in). Such counseling can become little more than affirming whatever someone feels, instead of speaking with authority to a person’s sorrows and sins. Puritan casuistry would make counseling more biblical. It would also display more of God’s authority and power, for He not only sympathizes, but also commands, judges, and sets the captives free. A Puritan example of such casuistical counseling can be found in William Bridge, A Lifting Up for the Downcast.
TC: One application you draw out is “be a preacher of the Word not a prober of feelings.” Why is this so important? How can a pastor ensure he is doing this?
JB: Certainly a pastor should ask questions about a person’s experiences, actions, and circumstances. He would be a fool to speak before he listens. But the pastor must never allow the world’s expectations of unconditional positive regard (or his own desire to please people) to make him into a sycophant and flatterer. Rather, the pastor is a messenger of the Lord of hosts (Mal. 2:7). He must command people to repent and believe the gospel because the kingdom of God is coming (Mark 1:15). He should ask himself, “Do my preaching and counseling apply both the law and the gospel? Is Christ the substance of it and supreme over all? Am I preparing souls for judgment day?” The idea that just talking about feelings brings resolution to difficulties is deeply mistaken. Repentance and faith must be exercised in the details or little changes.
TC: If a pastor or anyone else wanted to be counseled by the Puritans on casuistry, what would be the best books to turn to?
JB: Beginners could start with Thomas Brooks, Precious Remedies against Satan’s Devices, a great help regarding specific cases of temptation, and, as I previously mentioned, William Bridge, A Lifting Up for the Downcast.
A broad view of Puritan casuistry for the Christian life may be found in Robert Bolton, General Directions for a Comfortable Walking with God.
More advanced readers who are ready to tackle seventeenth-century print would benefit from reading the classic Puritan text by William Ames, Conscience with the Power and Cases Thereof (digital or in print).
The summa of Puritan casuistry is Richard Baxter, The Christian Directory (out of print, but available in digital format), but one must read Baxter with watchfulness because he is in error in his views of the atonement and justification.
I highly recommend the regular reading of Puritan sermons such as those by Thomas Manton on Hebrews 11. One can learn a lot about biblical counseling simply by paying special attention to the applications (“uses”) in each sermon.
If you are reading along with us, be sure to read Chapter 58 (“Puritan Sacrificial Zeal”) by next Thursday. Then simply check in here to see what Dr. Beeke has to say about it.
The purpose of this project is to read classics together. Please feel free to leave a comment below or to provide a link to your own blog if you have discussed this week’s chapter there.

A La Carte (10/25) [challies.com - Informing the Reforming] 2013-10-25 06:23
Lives Destroyed - “The past two years have been devastating. I have watched the lives of four Christian friends destroyed for want of care. These are men, I have loved and respected. All of them had families, loving wives, and children. Three of them were pastors and another was a nationally recognized professional at the top of his field. And all of them were consumed by their lusts.”
A Slow Learner - Julian is a slower learner and share some of the reasons he knows that.
$5 Friday - Ligonier Ministries has some good items on sale today in their $5 Friday: Sproul’s commentary on Acts along with books by Steve Lawson and Michael Haykin.
Jennifer - This man photographed his wife as she battled cancer. “Angelo decided to photograph it. He wanted to humanize the face of cancer on the face of his wife. The photos speak for themselves.” Indeed. It’s a tragic photo essay that speaks to a sin-stained world.
7 Perspectives on Prayer - Randy Alcorn provides seven short and sweet perspectives on prayer.
The Command to Kill - R.C. Sproul Jr. takes on this question: “Why did God command the children of Israel to kill every man, woman and child in the Promised Land?” Here is sound counsel: “Among the countless nuggets of wisdom I have received over the years from my father is this bit of gold- when you are reading your Bible and you come across something that makes you uncomfortable, resist the temptation to simply move on to something else. Where the Bible makes us uncomfortable is precisely where we need to slow down.”
The Elephant Man - You’ve probably heard of Joseph Merrick, aka “The Elephant Man.” Here an actor reads an Isaac Watts poem that was a favorite of Merrick’s while you see what the man looked like. Doctors still have not conclusively determined the nature of his condition. (See here to learn how they found what his voice would have sounded like.)
God is working out his eternal purpose, not only in spite of human and satanic opposition, but by means of them. —A.W. Pink


Mark Driscoll's Call to Resurgence [challies.com - Informing the Reforming] 2013-10-24 09:14
You may love him, you may hate him, but you’ve definitely heard of him and you’ve undoubtedly got an opinion about him. Mark Driscoll is pastor of Mars Hill Church in Seattle, co-founder of the Acts 29 Network, and the author of several bestselling books. The newest of these books may well prove the most controversial. A Call to Resurgence poses this question: Will Christianity have a funeral or a future? This is a book about the past, the present and the future of the Christian faith, particularly in the United States of America.
The fact is, Christendom is dead. The Christian faith that once existed in the background of American life and culture has diminished to such an extent that America is now a post-Christian nation. “Christians are ostracized. Gay marriage is celebrated. Abortion is literally destroying an entire generation. The bandwagon has stopped carrying us and has started running over us.” This is happening all around us, yet many Christians remain oblivious. “The church is dying, and no one is noticing because we’re wasting time criticizing rather than evangelizing.”
But, says Driscoll, this is not the time to debate. This is not a time for retreat but for resurgence. “We’ve got work to do. There are lost people to reach, churches to plant, and nations to evangelize. Hell is hot, forever is a long time, and it’s our turn to stop making a dent and start making a difference. This is no time to trade in boots for flip-flops. The days are darker, which means our resolve must be stronger and our convictions clearer.” “This book is an unflinching look at what we’re up against and what it will take to not just survive but to thrive and accomplish the mission God has given us to extend a hand of rescue to those drowning all around us.”
Driscoll believes he has been providentially situated to help the church in this time. He has spent the last twenty years of his life pastoring a church in the mission field of Seattle and believes that what was once unique to that city is now true of the entire nation. “I am convinced,” he says, “that what my church has seen become normative in our city will soon become normative elsewhere. The tsunami of cultural change hit our beach first, which puts us in a position to help others learn from our fruit and our failure. Maybe our little church plant was like Noah’s dove, sent to explore the landscape of a new world.” Thus he regards A Call to Resurgence as a kind of prophetic book through which he can lead the church in her response to these new realities.
Let me give a flying overview of the book to show how he builds his case and what he proposes as a solution. I will then share a few of my observations.
In chapter two, titled “Standing Knockout: How We Got Our Bell Rung,” he provides a brief history of evangelicalism. He traces its decline to Christians happily existing by themselves in their own little enclaves without seriously engaging the world, without earnestly evangelizing, and with far too much bickering with other Christians. He looks at some of the cultural forces that have pummelled Christianity with a series of hits: New Paganism, homosexuality, pornography, intolerant tolerance, bad dads, and cheap Christians.
Chapter three is titled “A New Reality: From Modernism to Everythingism to Tribalism.” With a nod to Seth Godin, he shows how tribalism is now more prominent than denominationalism and describes some of the common tribal commitments within Christianity. He highlights four questions and shows how Christian tribes may be distinguished by their answers to these questions.
He then builds profiles based on the possible answers. So, if you are Reformed, complementarian, cessationist and fundamentalist, “you probably like Together for the Gospel, the Gospel Coalition, Nine Marks, R.C. Sproul, reading books by dead guys, expository preachers who wear suits and have bad bands at their services, and wish this book had more footnotes and fewer jokes.” Driscoll himself is a tribal chief within the Reformed, complementarian, continuationist and missional tribe. “You probably have an occasional bad attitude, tattoo, impressive theological library and liquor cabinet, ESV Study Bible, entire collection of the latest indie rock, flannel shorts, and boots for no reason as you do zero logging.”
While tribalism is an unfortunate reality to life in a sinful world, it also represents a great danger to the church because it tempts us to fight one another instead of fighting for gospel advance. For Christians to continue to make a mark in this world, we need to avoid the extremes of “theological hard conservatism (which fights for too much) and theological hard liberalism (which fights for too little).”
Chapter 4 explores how tribes can hold to their convictions and still work together. He provides a picture based on geography.
So for Driscoll, Mars Hill is his home; like-minded churches are his neighborhood; the distinctives of Reformed, complementarian, continuationist and missional form the borders of his state; his region is other evangelical churches that love Jesus, believe the Bible and are led by Bible preaching; and Protestant Christianity is his nation. He can and will work with anyone inside that nation, even when there is loving disagreement on issues like Arminianism, egalitarianism, cessationism, and/or fundamentalism.
Of course for this to be effective, there needs to be agreement on the “national boundaries,” the doctrines that unite us by separating true believers and true churches from those that are fraudulent or outright heretical. Here Driscoll puts forth thirteen theses, each of which has to do with a specific doctrine.
Chapter 5 is titled “The Holy Spirit: Empowering the Church For Mission.” Driscoll is convicted about maintaining the unity of the Spirit and believes that unity of the Spirit requires unity about the Spirit. He provides a brief theology of the Holy Spirit that is consistent with his continuationist convictions. He affirms the existence of the miraculous gifts and gives a fair bit of attention to speaking in tongues, acknowledging the validity of this gift, but saying he does not have it. The main focus of the chapter, though, is giving attention to the Spirit’s role in empowering evangelism and bringing about conversion. He says, “Any truly Spirit-empowered ministry is not marked primarily by private spiritual experience but rather by the public proclamation of the gospel that leads to the salvation of sinners.”
Chapter 6 deals with “Repentance: A Biblical Response.” Here he revisits the cultural issues that have rung the church’s bell (see chapter two) and suggests how we can repent of these, not simply admitting our faults, but making real and lasting changes. In every case, Christians can go to the Bible and learn how we can stand out from the world around us.
The seventh and final chapter, “Mission,” offers seven principles for resurgence. “As we seek to encourage the preaching of the gospel for, the planting of churches by, and the discipling of the Gentiles of our day, there are some missiological principles that have proved helpful in Seattle.” He warns that these are principles, not methods, and provides a stern warning against “methodolatry.” They are:
He closes the book with one more call to see before us an unprecedented opportunity for mission. “Christendom may have died, but in that death there is a real opportunity for a resurgence of biblically faithful, personally humble, evangelistically fruitful, missional Christianity.”
There is much to agree with and to commend in A Call to Resurgence.
I appreciate his desire to waken a slumbering church. We are, indeed, in a new age and Christianity’s influence has clearly waned. What we once took for granted, what we once assumed, we no longer can. Too many Christians have missed this great shift in society as they’ve remained safe inside their holy huddles. Driscoll’s desire to see the saved awakened so we can see the lost saved is commendable. I appreciate his clear and bold calls to action.
I appreciate his emphasis on tribalism. While I hope we eventually come up with better terminology than “tribe,” I agree that denominations are fading in favor of less formal and more fluid relationships built on common commitments to secondary matters. I believe this tribalism is a product of this new, digital world. Whether it is a good or bad development will have to be a topic for another day.
I appreciate his desire to promote gospel agreement and gospel advance between tribes. We are bound by Scripture and conscience to hold to our convictions, but we also need to understand that secondary convictions must never become primary. We are always fighting the tendency to flee into our safe little enclaves and to be defined more by what separates us than what unites us. There are ways in which we can and must cross boundaries in order to help one another as we carry out our God-given mission.
These are just a handful of the many commendable qualities of A Call to Resurgence. I also have a few concerns, but will dwell on the two most prominent.
I am concerned about his criteria. Driscoll calls for Christians to work together despite their tribal loyalties. Because there are tribes that are opposed to the gospel, we need to consider the ones we can work with and the ones we cannot. Here Driscoll provides seven black-and-white issues that must be in place for a tribe to be considered evangelical:
I believe these criteria are too broad. Sound theology demands nuance, and that nuance is missing in these broad descriptions. Defining Trinity as “one God in three persons” still permits error and outright heresy. It is noteworthy that Driscoll’s list of Christian tribal leaders includes Joel Osteen, Joyce Meyer, Paula White, Joseph Prince, and T.D. Jakes (whom he has met and commends as a humble, Christian leader). I am not familiar with all of these people, but am comfortable stating that at least Joel Osteen and T.D. Jakes are false teachers who teach blatant, damnable error. Yet they would also, I believe, affirm the criteria he sets. T.D. Jakes can adhere to the heresy of modalism and still affirm that he believes in one God in three persons.
The criteria Driscoll uses to distinguish true from false Christians are simply insufficient. Either they need to be expanded, or we need an accompanying list of denials to go along with the affirmations. Any methodology that rates the difference between John MacArthur and Joel Osteen or R.C. Sproul and T.D. Jakes as a secondary issue between brothers is more than a little troublesome.
I am concerned with too little emphasis on discernment. I am concerned that A Call to Resurgence quietly commends those who run quickly to the pursuit of unity and quietly condemns those who hold back in an attempt to exercise cautious discernment. I have often observed that we are prone to criticize those who are careful and cautious discerners far more than we criticize those who pursue a reckless unity. Unity is a glorious quality and one we need to pursue, but we must not do so at the expense of discernment. And maybe we saw a glimpse of this in Driscoll’s actions last week, appearing at the Strange Fire conference—sponsored by John MacArthur and opposing charismatic theology—and passing out his own book there before commenting on social media that his books had been confiscated (something the evidence seemingly contradicts). His calls for maturity and unity appear to clash with his own actions.
Holding these two concerns together, I genuinely appreciate Driscoll’s desire to have Christians work with others beyond their tribe, but I believe he errs too far in the other direction and neglects to call out some false teachers and neglects to give clear criteria that will allow us to do so. His desire for unity is commendable, but even while we reach across the aisle to some we will need to be willing to unflinchingly reject and condemn others. I can hardly overemphasize the danger that may come here. I believe wholly in the value and necessity of working across tribal lines, but we must never do this at the expense of the primary doctrinal issues of the Christian faith.
A Call to Resurgence appears to find Mark Driscoll in a kind of transition. He has gained a lot of authority and notoriety in a relatively thin slice of the Christian world. It seems that he is now assuming leadership of a different kind, widening his scope to speak to all evangelicals. If they listen, they will hear much that is good and valuable and much that will spur them to greater action. Unfortunately, A Call to Resurgence could have been a much stronger book had Driscoll given more attention to careful discernment and the necessity of committing equally to truth and love.
(Note: This review was prepared using an Advance Reader Copy provided by the publisher; they warn that content may not be final.)

A La Carte (10/24) [challies.com - Informing the Reforming] 2013-10-24 06:01
Where Did All These Calvinists Come From? - Last week, Mark Dever dusted off a 2007 series he wrote on the New Calvinism and delivered it, with a few changes, as an hour-long lecture at Capitol Hill Baptist Church in Washington, D.C. Matt Smethurst provides a quick overview.
4 Reasons the Gospels Could Not Be Legends - “The most popular theory today against the Bible is that the gospels are a bunch of myths and legends. As the theory goes, Jesus was a great guy with some commendable teachings, but the stories we have about him in the four gospels are made-up legends intended to beef up Christianity’s claims.” Here are 4 reasons that just can’t be the case.
Secret Sexual Sin - The Lies Young Women Believe blog asked Aileen and me to write a couple of articles on masturbation—a growing concern as they provide counsel to young women.
Greater Work - R.C. Sproul gives an answer to this question: What did Jesus mean when he said we would do greater work than He did?
How to Write Less Badly - Michael Munger has ten tips on how to write less badly.
Jesus Calling - Christianity Today has a long and interesting article about the Jesus Calling phenomenon and its mysterious author, Sarah Young.
Strange Fire: What Now? - The GTY staff has a blog post about the aftermath of the Strange Fire conference. They include some words for the people who felt hurt by the conference and some encouragement as well.
Winners of souls must first be weepers for souls. —C.H. Spurgeon


Web Stuff Wednesdays [challies.com - Informing the Reforming] 2013-10-23 15:07
Greetings from your friends at Church Plant Media! We are back with another response to the questions posted to Tim’s blog in September. Joanna commented, asking us the following:
My question is how do you graciously but persuasively make a case to church leadership that a reasonable quality website is really important? Our website has out of date info, mismatching colors and the design looks like it came out of the late 90’s/early 2000’s. I’m concerned that it is alienating people, especially young adults newly arrived in the area to attend college, who are googling to find a church in the area. I don’t want to give the impression that the website is a magic bullet to anything but I do need to work out how to appropriately explain the impact of website quality on people trying to find a church to people who are less embedded in the digital world.
Joanna: This is a great question that we get asked from time to time. We all hope that people will be looking for a church where the gospel is preached every week, regardless of what the church website looks like. However the unfortunate reality is that many people still do “judge a book by its cover” before they decide to open the pages to see what’s inside. For most churches, their website is the “functional” front door before people step foot through the “literal” front door. This reality can be hard to understand if church members live most of their life unplugged. “Out of sight, out of mind” is the hurdle that you have to overcome.
You can overcome this communication barrier by using word pictures that the unplugged church member can understand. A helpful illustration that we have encouraged people to use is to compare the church website to the building where your congregation meets. Explain that most people will visit the church online before they visit the church offline. So if your church invests money into maintaining or improving the look and feel of your brick-and-mortar meeting space, they need to understand that an unkept, outdated website is similar to chipping paint, crumbling concrete, stained carpet, and a leaking roof on your physical building.
To put this illustration into practice, we can think about different parts of the church building and how they compare to the church website. If we can help you understand the website in the context of an online church building, you can help your unplugged members do the same.
These 5 items are important when considering a church building.
The cornerstone determines the position of the building. Its location is mission critical because every part of the foundation is positioned in reference to the cornerstone. The cornerstone of every church website should be the gospel of Jesus. When you clearly articulate your belief in the life, death, and resurrection of Jesus as the sinless substitute for sinners like us, you are positioning the cornerstone of your online ministry presence. If your website does not share the gospel, then that is where we suggest you start. When you get the gospel right, everything else will be able to line up accordingly.
The foundation needs to be big enough for the building. It needs to be solid, dependable, and functional. You can only build the building as big as the foundation will allow. The foundation of the website is the Content Management System (CMS). The CMS is the tool you use to build and maintain your website content. It is the backbone of the website that supports the online building. The CMS should allow you to grow your website no matter how big you get. If you have a faulty foundation, it can be difficult and costly to repair in the future. In the same way, be careful when you select a CMS to uphold and power your website.
The floorplan maps out every room within the building. Every congregation needs a type of “sanctuary” where the sermons are preached, a room where church events take place, and an area for additional teaching and gospel instruction to be provided. Just as a church needs these places in their constructed facility, they should also be made available on the church website. The sermons need to be accessible via a podcast and sortable in a sermon archive by date, series, and speaker. Events need to be accessible in an integrated church calendar. Additional teaching and announcements should also be made available on a church blog.
The exterior is the outer expression of the building. It is what people see before they walk through the front door. Some churches have steeples and siding, others have brick, mortar, and concrete, while others might have a renovated store front. The same concept applies to your website. In the same way the physical walls must be sturdy, a great website design must be more than just skin deep. Your church website should be built to address both search engine visibility and the design preferences within your church culture.
The entrance is the first impression of the building. Whether you call it a lobby, foyer, or narthex, this is the room that people experience when they first walk through the front door. Many churches will have people stationed at the entrance with bulletins and a welcome hand shake near a welcome center for new visitors. The home page of the website should function very similarly to your church entryway. Its purpose is to help answer the main questions of who, what, when, where, and how. Just as you think about greeting new visitors and introducing them to your church, you should also give thought to your website visitors.
These essential “building blocks” (cornerstone, foundation, floorplan, exterior, and entrance) should help any unplugged church member better understand the plugged-in church visitor. If you need other creative ideas on how to articulate your online church needs, do not hesitate to give us a call.
Your friends @ Church Plant Media | (800) 409-6631 x 1

18 Things I Will Not Regret Doing With My Kids [challies.com - Informing the Reforming] 2013-10-23 07:52
Like most parents, I have those moments where guilt and regret comes over me like a wave. I consider then how much of my parenting time has already passed by and how little remains. My oldest child, my son, is thirteen. He is already a teenager, just one year away from high school, just eight years from the age I was when I left home to get married. My girls are following close behind him. When that wave rises up, when I feel like I could drown beneath all that regret, I sometimes consider those things I will never regret.
Here are 18 things I know I will not regret doing with my kids.
1. Praying with them for them. It baffles me that one of the things that most intimidates me is praying with my kids. I don’t mean praying with the whole family before or after a meal, but praying with my daughter for my daughter or with my son for my son. Yet this kind of prayer lets them see that I am concerned for what concerns them and it lets us join in prayer together for those very things. I know I need to prioritize this because I will never regret praying with them for them.
2. Reading books to them. As summer turns to fall, as the days grow shorter and the nights grow colder, we spend many of our evenings together in the living room as I read books aloud. We’ve read our way across this world and across many others; we’ve read forward in history and we’ve read about days long past; we’ve met heroes and villains; and we have experienced it all together as a family. I will never regret reading books to my children.
3. Kissing them goodnight. The days get long and I get so weary. By the time the children head to bed I am sometimes so worn down that the very last thing I want to do is see the kids to bed and to kiss them goodnight. But I am always glad I did and often find these the times where the children are most tender, most eager to speak, and most eager to listen. I know I will never regret all those goodnight kisses.
4. Taking them to church. There is such joy in sitting in church together as a family, worshipping the Lord together and hearing from him in his Word together. I do not take my children to church so they can learn good manners or be better people; I take them to church so they can learn who they are, so they can learn who God is, and so they can encounter and experience Grace. I will never regret prioritizing church.
5. Taking them out for breakfast. One much-loved tradition in our family is taking my children out for breakfast on Saturday mornings—one of them each week. It’s a tradition I have lost and revived and lost again and revived again. It is a tradition worth maintaining. The $10 or $20 expense and the time it takes pales in comparison to the investment in their lives. I will never regret our breakfast daddy dates.
6. Letting my friends be their friends. I love it when my children befriend, and are befriended by, my friends. I want my children to have friends who are older and wiser than they are and friends who can help them in those areas where I am weak. I will never regret encouraging my friends to be their friends.
7. Doing family devotions. Family devotions is a difficult discipline to maintain, and especially as the kids get older and have more lessons and responsibilities. But we commit and re-commit and persevere because these are precious times—just a few minutes together to read the Bible, to talk about what we’ve heard, and to pray. I know I will never regret a single moment spent pursuing the Lord together.
8. Disciplining them. I hate disciplining my children; I hate having to discipline them. Yet I am absolutely convinced that to refuse to discipline them is to refuse to love and respect them. The lost privilege, the stern talking-to, the time spent alone in their room—these are all seen as hatred in the moment, but seen as love later on. I will never regret lovingly disciplining my children.
9. Doing special things. Life is largely lived in the mundane and love is mostly shown in the day-to-day. But there is also value in the afternoons at the ballgame, the evenings at the ballet, the business trips with dad. I will never regret doing those special things with my children.
10. Asking their forgiveness. I have more trouble apologizing to my children than to anyone else. Somewhere way in the back of my mind I am convinced that to apologize to them is to show weakness; but at my best times I know that to apologize to them—to ask their forgiveness when I have sinned against them—is honoring to God and to them. I will never regret those times I have asked their forgiveness.
11. Forgiving them. My great weakness is one of my kids’ great strengths; when they sin they are almost always quick to seek my forgiveness. I will never regret sincerely and immediately granting the forgiveness they ask.
12. Loving their mother. I know that the stability of a mother and father who are firmly committed to one another brings stability to the whole family. I can love my children by assuring them of my love for their mother through my words and deeds and affection. I will never regret regularly affirming my love for their mother.
13. Identifying God’s Grace. As my children make professions of faith and as they begin to grow in godly character, it has been a joy to see God’s grace in their lives. I am learning to tell them what I am seeing, to commend them for it, and to point to the One who has generated it. I know I’ll never regret identifying this kind of grace in their lives.
14. Expressing affection. I love to walk hand-in-hand with my daughters and I love to hug my son before he goes off to school. This physical affection makes them feel safe and loved while teaching boundaries and platonic, appropriate touch. I will never regret continuing to express physical affection.
15. Planning little surprises. The small and occasional gifts when I return home from a speaking engagement; a single rose for my girls when I buy their mother a bouquet of flowers; the dinner at McDonald’s for no reason at all. I will never regret planning and delivering these special little surprises.
16. Giving them my full attention. I almost always have an electronic device within reach and often I have two or three of them. It is so easy to break out of a conversation with every buzz or beep, to break eye contact and to break concentration. I know I’ll never regret giving my children my full attention when they have something to say.
17. Pointing to the gospel. The gospel is not merely a gateway to the Christian life, but the very source of hope and joy in the Christian life. We need to return to the gospel again and again; we need the gospel every day. And I will never regret pointing my children to the gospel.
18. Telling them “I love you”. I love my children dearly and I can show that love in each of the ways I’ve listed above. But when they head off to school, when they go out with friends, when they call me at the office, when we FaceTime from afar, I will never regret telling them one more time, “I love you.”
What are some things you will never regret doing with your kids?
(With a hat tip to HuffPo)

A La Carte (10/23) [challies.com - Informing the Reforming] 2013-10-23 05:41
I heard from some of you that you had trouble with yesterday’s Kindle deals. Here they are again in case you weren’t able to take advantage: Magnifying God in Christ by Tom Schreiner ($4.99); A Little Book for New Theologians by Kelly Kapic ($0.99); Worship Matters by Bob Kauflin ($3.99); The Psalter Reclaimed by Gordon Wenham ($3.99); Rhythms of Grace by Mike Cosper ($3.99); Introducing Covenant Theology by Michael Horton ($3.99).
Being Better Bereans - Kevin DeYoung has begun a three-part series titled “How To Be Better Bereans.” It’s off to a good start with part one.
Why Is Faith Not a Work? - “Why is faith not a work? If we are obligated to have faith before righteousness can be credited to us (Romans 4), how is faith not a work? I recognize that Paul tells us in Ephesians 2 that we are saved by faith not through works, but I don’t quite understand how to reconcile faith not being a work if we are required to have it in order to be saved.” Matthew Barrett answers.
My House Just Burned Down - “Joel Hollier is a student at Sydney Missionary and Bible College. He and his family lost their family home this week in the bushfires raging through the Blue Mountains of New South Wales. He posted this moving reflection on Facebook, and has given us permission to publish it here.”
Bouncing the Eyes - Men are taught to fight lust by bouncing their eyes. Mike Leake says, “That was the gist of how I was taught (and in turn taught others) to battle lust and pornography. While I do believe there is some warrant to this teaching, I believe now that it is largely incomplete.”
G3 Conference - This will be of interest to you if you’re thinking of attending the G3 Conference in Atlanta this January (Speakers: Voddie Baucham, Steve Lawson, Conrad Mbewe, myself and others). They are offering a special deal on October 31 where, for that day only, registration will be 2 for 1!
Russell Moore in WSJ - The Wall Street Journal recently profiled Russell Moore.
To know that nothing happens in God’s world apart from God’s will may frighten the godless, but it stabilizes the saints. —J.I. Packer


The Art and Science of the Humblebrag [challies.com - Informing the Reforming] 2013-10-22 08:07
Of all the words coined in response to the realities of this digital world, of all the words recently added to the dictionary, humblebrag must be among the best. According to the Macmillan dictionary, a humblebrag is “a statement in which you pretend to be modest but which you are really using as a way of telling people about your success or achievements.” It is bragging in the guise of humility, putting a thin veneer of humble over a clear expression of proud. And it seems to be an integral part of an effective social media presence.
Have you managed to get thousands of people to follow you on Twitter or friend you on Facebook? Do you need to keep reminding them why you are worthy of their attention? Let me offer you some ways you can grow in the art and science of the humblebrag.
Tell others what you own. The humblebrag is a great way to subtly tell other people about your cherished possession while at the same time dropping hints about your excellent financial situation. “When I bought this Ferrari no one warned me I’d get pulled over all the time.”
Brag about your opportunities. As your fame increases, you will inevitably be given more and better opportunities. Each of these opportunities offers the possibility of a humblebrag if you know what to do. “My fingers are aching from typing my memoir all day…”
Make sure they know who you know. Fame is contagious, you know. You can always elevate yourself in the eyes of others by cashing in on friendship or even just relationships with people more famous than yourself. Make sure the people who follow you know about every famous person you meet. “Bumped into my dear friend Tom Hanks at the Academy Awards tonight. He’s awesome.”
Remind them that you’re popular. The humblebrag is an ideal medium for quietly telling others about your popularity. “Preached the worst sermon of my life but still got a sore hand from signing all those Bibles afterward.” Or again, “I never get used to seeing my face up there on the billboards.”
Tell them about your charity. Humility often works itself out in good deeds, so you will need to be sure to let people know about some of the good things you do. “Guy who rents my apartment lost his job. Told him not to worry about rent this month. Paying it forward, baby.”
Have you mastered the basics of the humblebrag? Then you are ready to move into the advanced techniques.
Hide it in a question. Here’s another advanced technique: try hiding your accomplishment in the form of question. “Is anyone else going to be at the White House tonight? It would be great to meet up…” Or again, “Does anyone know if you can claim a yacht as a home office?
Declare your humility. Obviously you want people to know about the great things you’ve done, but they also need reassurance that you’ve stayed humble through it all. Here’s a great way to do it: Try beginning a social media update with the words, “Humbled that…” and follow it with your milestone or accomplishment. Example: “Humbled that my album hit the Billboard Top 100.” (Note: Some people will tell you that by its very nature humility does not speak of itself, but don’t let that stop you.)
Blame it on Jesus. The best humblebraggers know how to legitimize their boast by incorporating the divine. This way it’s not really about you, it’s about Jesus. Try this: “I just got asked to perform at the Dove Awards. Go Jesus!”
Feign embarrassment or awkwardness. You’re always humble when feeling awkward or embarrassed, right? You can feign it and marvel others with your humility. “That awkward moment when you ask Jim Gaffigan to sign a book…and he asks you to sign yours.”
GrumbleHumble. Try wrapping your brag in a grumble, using a complaint to let people know how awesome you are. “Tried shopping on Amazon and they recommended my own book to me. Fail!” “I hate it when you get profiled on 60 Minutes and they mispronounce your name.”
Bragging is an integral part of your social media presence. I trust this little guide will prove helpful as you humbly brag about all the good things you are, all the great things you have, all the excellent things you’ve done.

A La Carte (10/22) [challies.com - Informing the Reforming] 2013-10-22 06:20
Here are a few new and notable Kindle deals: Magnifying God in Christ by Tom Schreiner ($4.99); A Little Book for New Theologians by Kelly Kapic ($0.99); Worship Matters by Bob Kauflin ($3.99); The Psalter Reclaimed by Gordon Wenham ($3.99); Rhythms of Grace by Mike Cosper ($3.99); Introducing Covenant Theology by Michael Horton ($3.99).
You Might Be a Christian Celebrity If… - This may hurt. “In blogdom, writing, and pastoral ministry in general, there is a temptation to pursue being popular; to pursue being a celebrity. Yet, in order to follow Christ, we must deny ourselves, take up our crosses, and follow Him (Matt. 16:24). This includes denying our desire to be popular, our desire to be celebrities. Are you a celebrity Christian wannabe? You might be one if…”
Prodigal Sons - I always loved the Modern Parables series of short films. Today only you can download Prodigal Sons for free using the code PSJT. Do it. Do it now! (more information)
Murder for Hire - “I’ll be the first to admit it; hit men are shady. But they are shady because they are doing work that no one else wants to do, work that is, in fact, illegal. By labeling contract killing a ‘crime,’ we have obscured the fact that hit men provide a valuable service to society.”
Skull of Homo Erectus - I think it’s important for us to keep being reminded how often the story of evolution changes, and often by millions of years at a time.
For the Overwhelmed - Jen Thorn shares hope for the overwhelmed mom. “How in the world am I going to keep up with deadlines, clean up a home that has TOTALLY exploded over the weekend, homeschool and deal with all the other little things that will fill my day?”
7 Things About Calvin and Hobbes - Here are 7 things you may not know about the always-excellent Calvin and Hobbes.
A gracious soul may look through the darkest cloud and see God smiling on him. —Thomas Brooks


Lessons Learned at Strange Fire [challies.com - Informing the Reforming] 2013-10-21 09:14
When I began blogging through last week’s Strange Fire conference, I had no idea how big an impact the event would have. Even while attempting to transcribe John MacArthur’s opening address, I was not convinced I wanted to dedicate three days and eight or ten articles to it. But once I began to see and hear the reaction, I determined there would be benefit to listening in, writing it down, and in opening it up for conversation.
I attempted to make my summaries as objective as possible—simply sharing what each speaker had said without offering my own opinions. Today I want to circle back one more time to share a few final reflections on the event. Here is what I am thinking several days later.
This is a worldwide issue and I need to ensure I see it that way. We need to ensure we see it that way. Those who listened to the conference heard again and again just how many charismatics there are in the world—somewhere around 500 million. Conrad Mbewe made it clear that in many places in the world, and especially in the developing world, to be a Christian does not mean that you trust in Jesus Christ for salvation, but that you believe in and practice something akin to the miraculous gifts. Charismatic theology is a North American export that is making a massive impact elsewhere in the world.
There is a challenge here for myself as a Reformed, North American believer: I have a very narrow view of the Christian world—a too-narrow view. MacArthur made it clear that he did not host this conference in order to critique the Wayne Grudems and John Pipers of the world; if these men were representative charismatics, Strange Fire would have been a non-event or, at the least, a very different event. He hosted the event because there are hundreds of millions of people around the world who make the fraudulent practice of fraudulent gifts the heart of their expression of the Christian faith.
This is the time to address that issue. There is a call here for all of us to build on and even improve what MacArthur began and much of the onus here falls on charismatics to do this from the inside. As Clint Archer concludes, “All true believers are on the same team, and we’re all against the abuses and excesses of masquerading unbelievers. Conservative Continuationists need to start their own version of the conference to police the excesses as best they can, or they should muster a cheer while the Cessationists do it.”
The charismatic/cessationist issue is polarizing. Before Strange Fire I did not know just how polarizing it could be, though I suppose others did know, and this is why we have been loathe to address it. Based on the reaction to the event and the discussions back-and-forth, it seems clear that this is an issue many of us feel as much as it is an issue we believe by reasoning it out from Scripture. It is one of those issues where we see our own position with utter clarity and look to the opposite position with shock that they can believe something so absurd. Those tend to be the most dangerous issues of all because they can turn sour so quickly and easily. In the face of such a polarizing issue, I need to consider how I can maintain unity in the faith while still holding fast to what I believe the Bible teaches.
I saw at Strange Fire that we can sometimes confuse confidence with arrogance. And it’s not just we, but me because I suspect that if the tables were turned, I might react in much the same way. I am convinced one of the reasons so many people reacted badly to the event is that MacArthur and the other speakers are so sure of what they believe. They spoke with confidence about their understanding of what the Bible permits and what it forbids. Some of the reaction from those who were offended seems to imply that certainty is incompatible with humility. If this is what they truly believe, they have succumbed to dangerous and worldly thinking.
Trevin Wax goes into some detail on this and says, “If you agree with MacArthur, the best way to engage critics is not to defend him as if he were the pope, but to back up your claims by appealing to Scripture. If you disagree with MacArthur, the best way to engage the conference is not by railing against the man, but by showing specifically the ways you think he caricatured your position and by providing a calm, sober affirmation of continualist claims, backed up by Scripture.” And again, “let’s not judge the conference speakers as wrong simply for gathering together and taking a stand against doctrines they believe to be false. As Christians, we may be continualists or cessationists, but we are not relativists.”
I have long believed that many of the issues related to charismatic and cessationist theology owe to misunderstandings between the two sides. The reaction to this conference—the many discussions through social media and elsewhere—reveal that we need to do a better job of understanding one another, of affirming common ground, and of determining the importance of our differences. As a convinced cessationist, I was troubled to hear caricatures from charismatics about quenching the Holy Spirit, about elevating Scripture above God, about excluding all possibility of miracles, and so on. All of these caricatures show an uncharitable and unhelpful misunderstanding of cessationism. I am sure many cessationists were equally unfair and that I, myself, do not understand the continuationist position as well as I should. The simple fact is, until we rightly understand one another, we are in a weak position to bring critiques. But I know I am prone to do it anyway, to argue out of ignorance. I have to challenge myself here to be quick to listen and slow to speak, and when I do speak, to speak through the Scriptures.
This is a late addition to the article (a half hour after posting it), but I wanted to express it. We always face the danger of making our theology about who we believe rather than what we believe. The last thing we want or need is “I am of MacArthur” and “I am of Grudem.” I am sure this is the very last thing those men want. So even while we take our cues from the men we admire and the men who may think better than we do, let’s be sure that we are all Bereans, that we are all going back to the Bible to determine what we believe. Let’s be known for what we believe far ahead of whom we believe.
Strange Fire was an event that primarily targeted the worst of the charismatic movement. As I said when I offered an early look at the book, it is more about Benny Hinn than Bob Kauflin. While the Reformed charismatics may be a valued and significant part of the New Calvinism, they represent only the smallest fringe of the wider charismatic movement. What still remains to be done is to interact with the best arguments of the best of the charismatics and to address this from within the Reformed resurgence. This would be a very different event with a very different purpose and I hope someone will sponsor it before long.
Only time will tell of the long-term impact of Strange Fire, but as I think back to the past few days, I find myself grateful for it. I suppose that may be easier to say as a cessationist than a charismatic, but I believe the event and its aftermath will prove beneficial. I continue to pray that God would use it to to strengthen His church and to glorify His name.

A La Carte (10/21) [challies.com - Informing the Reforming] 2013-10-21 06:00
There should be new Kindle deals tomorrow, but for now, here’s a round-up of deals that are about to end: The Dragon’s Tooth by N.D. Wilson ($2.99); The Justification of God by John Piper ($3.03); Manhood Restored by Eric Mason ($4.74); The Pursuit of God by A.W. Tozer ($3.81); Putting Amazing Back Into Grace by Michael Horton ($3.49); Liberating Black Theology by Anthony Bradley ($2.99); Keep Your Head Up edited by Anthony Bradley ($2.99); Glory Road edited by Anthony Carter ($2.99); The Faithful Preacher by Thabiti Anyabwile ($2.99); The Baker (Non-)Illustrated Bible Commentary ($1.99).
Worship Him Well - I love to hear this: “I have walked the aisles of Notre Dame in Paris and of St. Patrick’s Cathedral in New York City. They are works of magnificent splendor. Stunningly beautiful, to say the least. But they did not come close to giving me the feeling I have every time I step into that loud and unaesthetic gym.”
Doubts: Fatal or Futile? - If you struggle with doubt, this article may be helpful. “These types of doubts, spiritual in nature, have the ability to unmoor a person. Doubts like these often serve as the doorway towards deeper issues like depression, paralyzing fear, and chronic anxiety.”
Wildlife Photographer of the Year 2013 - I’ve got a soft spot for photography competitions. Here are winning photos from this year’s wildlife photography competition.
Married Mom and Dad Do Matter - “A new academic study based on the Canadian census suggests that a married mom and dad matter for children. Children of same-sex coupled households do not fare as well.”
Cereal Transformed American Culture - I think they overplay the Christian fundamentalist angle at the beginning of the article, but it’s fascinating nonetheless.
Nearness to God brings likeness to God. The more you see God the more of God will be seen in you. —C.H. Spurgeon

The Philanthropists: R.G. LeTourneau [challies.com - Informing the Reforming] 2013-10-20 06:30
Robert Gilmour LeTourneau was an influential businessman and inventor of machinery that shook the world (since, after all, much of the earthmoving equipment used in World War II was made by LeTourneau’s factory). Born in Richford, Vermont in 1888, LeTourneau became a wealthy and generous Christian philanthropist who worked hard and shared freely.
With the reluctant support of his parents, LeTourneau dropped out of school in sixth grade to work in the mechanical field. In 1919, he married Evelyn Peterson whose father owned a company that built hay wagons. Throughout his lifetime, he lived in numerous places and practiced numerous vocations including cutting wood, mining, carpentry, farming, and, of course, mechanical work. Such a variety of experiences contributed to his impact and equipped him to leave a significant legacy.
God blessed LeTourneau with godly parents who shared the gospel with him from his birth. Although resistant for a season, he professed faith in Christ at the age of sixteen. Around the time he got married, LeTourneau told his pastor that he wanted to do more for God. Much to his surprise, his pastor told him, “God needs businessmen too.” At the time, he was $100,000 in debt because of a large-scale construction job that had gone poorly. But even though he was in the midst of such financial difficulties, he trusted God with his money, family, and life, knowing that God uses man’s weakness to showcase His strength.
LeTourneau became a leader in the Christian & Missionary Alliance Church through the numerous speaking opportunities he had in the US and overseas, and through his work as the president of both the Christian Business Men’s Committee (CBMC) and Gideons International (known for their vast Bible-distributing efforts). The fact that he had become such a renowned public speaker is a testimony to God’s enabling power in light of the fact that LeTourneau previously had a lifelong fear of speaking in front of others.
LeTourneau is known for living on 10% of his income and giving 90% of it away for the sake of God’s kingdom. Because of his belief in the power of hands-on training, LeTourneau founded the LeTourneau Technical Institute to provide a wide variety of instruction, from traditional college courses to technical and mechanical training. In fact, with his wife Evelyn’s help, his technical institute became LeTourneau University and together they founded the “LeTourneau Christian Center” in campgrounds in New York. His University and Christian Center served to multiply his influence throughout the world in the lives of young, passionate Christians who loved God and loved others.
By 1959, even after donating $10 Million to Christian and educational endeavors, the LeTourneau Foundation was still worth around $40 Million. LeTourneau suffered a stroke in 1969 at age 80. He left behind a legacy of great Christian generosity. In fact, because of his reputation for giving, even during his life his colleagues called him “God’s businessman.” Inscribed on his grave are Jesus’s words in Matthew 6:33, “Seek ye first the kingdom of God and His righteousness and all these things shall be added unto you.”

Weekend A La Carte (10/19) [challies.com - Informing the Reforming] 2013-10-19 06:43
Why You Should Care - Thabiti Anyabwile on Strange Fire: “An honest discussion on this topic entails a number of uncomfortable admissions. And, perhaps it’s our inability or unwillingness to admit these things that hinder our discussion as much as anything else. We feel the stakes but we don’t want to face the stakes. For some people, this conference forces some difficult admissions, admissions we’d be happier not to concede. But, that, too, is reason to care about the issue and engage prayerfully. Consider what’s at stake.”
A Theology of Book Purging - I quite enjoyed this: “A Concise Theology of Voluntary, Principled Book Purging.”
Comfort and Contentment - Darryl Dash wonders about the difference between being comfortable and being contented.
5 Things Students Should Know - Louis Betty is an assistant professor of French at the University of Wisconsin at Whitewater who recently wrote an article titled “5 Things You Don’t Have the Guts to Tell Your Students.” It’s worth a read.
Does God Listen to Rap? - I know some of you are fans of Christian rap, so I thought you might enjoy reading the first chapter of this book, which Cruciform Press will release on November 1.
Can I Vent? - “Have you ever asked someone that? Or been asked that? … We weren’t really looking for advice, we were just venting. Venting is not looking for counsel. It’s mostly just complaining.”
Iconic Goals - Here are twelve iconic World Cup goals and the stories behind them.
We complain today that ministers do not know how to preach; but is it not equally true that our congregations do not know how to hear? —J.I. Packer

Strange Fire Conference: MacArthur's Appeal to His Continuationist Friends [challies.com - Informing the Reforming] 2013-10-19 06:02
The Strange Fire conference closed with a final address from John MacArthur. In this address he responds to seven accusations brought against the conference, follows with eight appeals to his continuationist friends, and concludes by walking through 1 and 2 Timothy, highlighting the need to stand firm in guarding divine revelation against false doctrine.
Before addressing the accusations against the conference, MacArthur charged attendees to carefully read their copy of Strange Fire and to measure it against the Word of God. He is convinced that this book, with its well-documented research and extensive footnotes, will withstand careful scrutiny. He reminds us that this book and conference is intended for the Church. He has no expectation for either one to be helpful to non-believers, which he suspects makes up much of the charismatic movement.
MacArthur then shared from his heart responses to seven accusations against the conference. These accusations have arisen from the Internet. It is interesting to note that we live in a time where we are able to give more people access to information simultaneously like never before, which then puts us quickly under scrutiny as never before.
Here are the seven accusations, along with brief responses.
MacArthur remarks that this is an alien movement springing forth from the 60’s hippie drug culture. It is seeker friendly and culturally driven. The heroes of the charismatic movement are fundamentally different than ours. If you are a cessationist, then you have a responsibility to police this movement. Therefore, he wrote an open letter to his continuationist friends to re-examine their position. These are real friends of his—people he respects, has ministered alongside, prayed with, and hammered out convictions with. They are doctrinally conservative pastors and scholars that call themselves continuationists because they want to give place to this movement.
MacArthur provides 8 statements to why they must help us.
MacArthur argues that the charismatic movement and the position of continuationism opens the door to more theological error than any other doctrinal aberration which has preceded it. The true church needs to respond. These errors must be corrected. Don’t call yourself a charismatic Calvinist. Calvin rejected that himself. If you insist on staying in that movement then drop the label of Calvinism. If these theologically conservative continuationists take a turn to cessationism they may make a massive difference for the next generation.
MacArthur closed the conference by walking through 1 Timothy and 2 Timothy. He emphasized the importance of Timothy guarding divine revelation and protecting it from empty chatter and false teaching. In 2 Timothy this is a matter of great concern for Paul. He worries that Timothy wavers on the edge of cowardice. So again and again Paul exhorts Timothy to be strong, take a stand and fight the good fight. We are encouraged by a brief word in Hebrews 13:23 where we discover that Timothy has been released from prison. Apparently Paul’s parting words gripped his heart. He mustered courage, spoke the Word boldly, guarded it from false teaching, and went to prison for his conviction. Paul’s letter changed the course of Timothy’s life.
Paul stood alone towards the end with few friends: Onesimus, Luke, and Mark counted as them. Yet he confidently was able to bestow the ministry of the gospel upon Timothy. MacArthur is thankful that wonderful people surround him. He cannot say that he has been forsaken quite like Paul, though he has been vilified as will anyone who stands firm on issues like this.
May we guard the treasure, retain sound words, study to show ourselves approved unto God, workmen needing not to be ashamed, rightly dividing the Word, and preaching that Word. And if no one stands with us: so be it. The Lord will strengthen us.

Strange Fire Conference: Preachers or Witch Doctors? [challies.com - Informing the Reforming] 2013-10-18 14:57
The first session of the final day at the Strange Fire conference brought Conrad Mbewe back to the pulpit. Phil Johnson introduced him by sharing how others have called him the Spurgeon of Africa. Today he brought message entitled, “Are We Preachers or Witch Doctors?”
Though an odd question, it is pertinent to him because there has been a clear shift in how “evangelicals” relate to pastoral ministry. Mbewe’s aim is to give a broad sweeping picture of the landscape of African “evangelicalism.” Throughout this message his caveat is to put “evangelicalism” in quote and end-quote, because it does not represent biblical and faithful Christianity. There will be those in Africa who do not fit within the picture Mbewe portrays, but what he shares today is the trend and it is a dismal trend.
2 Timothy is the last epistle Paul writes because he will soon end his labor and depart from the world. This is not the end of the Christian Church. This exhortation is Paul’s parting gift to Timothy, much like Christ’s high priestly prayer is a departing gift to the Church.
First, this exhortation in 2 Timothy 3.16-4.5 illuminates how Scripture is a sufficient tool to accomplish all the work the man of God is to do. He is adequately equipped by the Word of God. Second, this is a charge to Timothy, a hair raising charge, evoking God the Father and the Son to preach the Word. Third, once an audience is found, even if it has stuffed ears, Timothy is to do all he can to ensure that the Word is preached.
Mbewe then contrasts Paul’s exhortation in 2 Timothy with the present picture in Zambia. He shares two newspaper clippings from July concerning evangelical preachers. In the first, a clergyman impregnated 10 women before his wife came forward about the scandal. She came forward after witnessing the scourge on the children in this church and the presence of the witchcraft taking place. In the second clipping two clergy men took two women into the mountains and sexually assaulted them. They first visited their home and took prayer requests and then led them into the mountain.
Conrad asks, “How can this be happening so frequently among so called evangelical churches today?” His response: a seismic shift in how people view the pastor. What is read in 2 Timothy is not the popular view today. It is because of how the view of the “man of God” has evolved today. Pentecostalism’s visit to Africa did not primarily emphasize the preaching and teaching component of the “man of God.” Now the “man of God” is primarily seen as the deliverer. Preaching has lost emphasis. It has become motivational platitudes followed by shouting and chanting.
The same thing can be seen in America on popular TV channels, but with different colored skin and nicer buildings. There are biblical quotes tossed about followed by a demon possessed, crazy and maddened looking preacher.
Yet, the important part of any service is what happens next. The “man of God”, wrested out of the context of 2 Timothy, takes on the role equivalent to a witch doctor.
Timothy was told to preach the Word of God—what is described as teaching, correcting, training, and rebuking in righteousness. Yet, back home, people are waiting for the point when hands are laid upon them and they are delivered. They are waiting for the “man of God,” wreaking with power to do his work. This is after a prolonged evening prayer meeting going into the night followed by an all day service in the mountains, just like in the clippings. Remember, the picture of the mountains in the clippings. This is what takes place in the mountains!
Sadly there is not effort towards biblical counseling. A man without work is not asked about his work ethic. A marriage in jeopardy is not examined to see if the husband loves his wife like the church or the wife submits to her husband. In America Mbewe has heard the radio preachers do likewise. Rather than counsel on the radio they invite the caller with question about infertility to come to a prayer meeting and seek deliverance. This is the penicillin for all issues. No moral questions are asked. This is precisely what the witch doctor does.
First, he claims spiritual discernment after lengthy time in prayer. He has secret dealings with God. Then he tells you what your real problem is. He goes into a trance, moves pebbles around, and announces that your neighbor has spoken to the spirit of your dead uncle who is obstructing your pregnancy.
Second, the witch doctor seeks to obstruct conventional medicine, which function as a threat. If you betray his trust you are undoing what the “man of God” has done by going to the hospital for the white man’s medicine. Many have died of illnesses that could easily have been prevented had they come earlier for treatment. The markings on their bodies betray what has happened and the work of the witch doctor. With that pastor, “the man of God’s” mark is left on the family who can tell why the person delayed in visiting the hospitals.
Mbewe repeats the question: How can this be happening so frequently among so called evangelicals? The people keep their lips zipped because of ignorance and because of blind loyalty in frauds. “The man of God” is the equivalent of the witch doctor. “The man of God” has an eerie connection with God where lesser mortals do not. This is not the priesthood of believers. Exposing “the man of God” brings a curse on oneself. Therefore, the people pretend to know nothing.
Consequently, when “the man of God” does what borders on immoral, there is no premise to blow the whistle. Mbewe is shocked by the number of women who have this story and fail to come forward just like the woman in the newspaper clipping. He wishes this was just about cows or some traditional African religion. But this is the new face of African evangelicalism to the outside world. It is a far cry from 2 Timothy 3.16-4.5.
“The man of God” will be dressed in flashy dress like a worldly pop idol with a feigned American accent. Their sermons are not worth listening to twice. There is no exposition of Scripture and no display of the unsearchable riches of Christ. Repentance is conspicuously absent. There is no effort to conform the people of God to the image of Christ. Rather, it is about how you get so much to spoil yourself. One statement that represents “the man of God” is this. “I declare prosperity on your life in Jesus name.” This irreverent invocation repeated is just like the witch doctor with his incantations. In reality nothing really happens!
Mbewe was invited to a radio broadcast panel discussion in Zambia about miraculous healing. There was a Catholic trying to ride the fence. Then there were two charismatics invited. One could not come because he was sick. He lied. Conrad saw this man shopping in the mall with his wife directly afterward with a trolley of goods.
During the broadcast the other charismatic and Mbewe locked horns. He challenged listeners to call in if they had been healed. Like a New Testament Elijah he taunted the charismatics for an hour due to the lack of calls. Two calls came in. The first a man who attested to a girl with unequal legs being healed 8 years ago, a very stale testimony for a country that claims to have healing crusades from prophets, bishops and “the man of God” all the time. The second came from a woman who chastised Mbewe as a dead theologian. There were only two calls in a nation where miraculous gifts happen all the time. The charismatic pastor responded that the people are shy. Unfortunately a week later he suffered a stroke and died after being in a coma for a week. None of his friends came to his aid and raised him because they knew it was all a fraud and a lie.
Mbewe shared that Zambia has an organization called the council for the handicapped, which revealed that not one actual healing has ever occurred. The council instructed people to stop going to these deliverance meetings.
He closed by apologizing. He apologized for bringing this dismal report in the morning as everyone enjoyed his or her coffee and donut. He wishes this was happening in a fringe corner of the world. Rather, the modern day successful African evangelical pastor is but a witch doctor using clearer and more potent power to bring about deliverance and breakthrough for the people. In light of 2 Timothy, he is concerned about the silence in addressing this matter. His blog post, “Our Criminal Evangelical Silence” was written because it bothered him. 90% of African evangelical fellowships are members of these kinds of ministries.
Mbewe’s final appeal is first, if you go to Zambia or Africa, call the pastors back to what their work is as designated in 2 Timothy. What sets apart a congregation is not some powerful connection to God to connect and talk to spirits. It is the calling to preach the Word of God. Second, pray and keep praying for those who are fulfilling 2 Timothy 4.1-2. Pray that they will be faithful and increase in number. Pray that the word of God will bring light into the darkness. Third, provide truth especially through books that clarify this issue, sound a warning, and give a clarion call to faithful exposition of scripture. Last support the training and work of true preachers and church plants that represent New Testament Christianity.

Free Stuff Fridays [challies.com - Informing the Reforming] 2013-10-18 09:07
This week’s Free Stuff Fridays is sponsored by Crossway and they are offering a selection of brand new books for you. They have five prize packages to give away and each of them will contain the following three books:
Joseph and the Gospel of Many Colors by Voddie Baucham. “We think we know the story of Joseph—the young man sold into slavery by his own brothers before rising to immense power over all of Egypt. But is it possible that we’ve missed the real story behind the story? In Joseph and the Gospel of Many Colors, Voddie Baucham Jr. helps us to understand the crucial role that the story of Joseph plays in redemptive history. Engaging and thoughtful, this book will help you read the Bible from a Christ-centered perspective and revitalize your love for God—the true hero of history.”
What Is the Meaning of Sex? by Denny Burk. “Sex. We live in a world that loves it without understanding it. This book clearly explains the truth about sex and winsomely responds to society’s evolving views on human sexuality and gender. From marriage to birth control, homosexuality to singleness, What is the Meaning of Sex? sets forth a distinctly Christian perspective, equipping you to engage our confused culture with a God-glorifying vision of human sexuality.”
Found in Him by Elyse Fitzpatrick. “Everyone, Christians included, knows what it’s like to feel isolated and alone. We’ve all wondered if anyone really understands us or truly cares about our lives. The good news is that we aren’t alone, and the gospel tells us why: Jesus, the Son of God, came to earth to be forever united with his people—to be one of us.”Giveaway Rules: You may enter one time. As soon as the winners have been chosen, all names and addresses will be immediately and permanently erased. Winners will be notified by email. The giveaway closes Saturday at noon.
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Strange Fire Conference: Scripture Alone [challies.com - Informing the Reforming] 2013-10-18 07:14
The final session on day two of the Strange Fire conference was led by Steven Lawson who spoke on “The Puritan Commitment to Sola Scriptura.” This was another historical message meant to demonstrate how our forebears were committed to the doctrine of Scripture alone.
Tonight the focus of our study will be another historical theology overview of a critical issue that ties in wonderfully with this entire conference.
It’s the subject of the Puritan commitment to Sola Scriptura. Sola Scriptura (Latin for “Scripture alone”) served as the foundation for the four other solas of the Reformation: sola gratia (by grace alone), sola fide (by faith alone), solus Christus (Christ alone), and soli Deo gloria (glory to God alone). These five all fit together as one statement of truth, one declaration of the true saving gosepl of Jesus Christ. Sola scriptura is the foundation upon which everything rests, everything we believe, embrace, and hold dear.
Rome said “We accept Scripture, but also Church tradition, ecclesiastical hierarchies, etc.” But the Reformers said “No, it’s sola scriptura. If anything else is added to the foundation of the church, the foundation will be split and unable to hold the rest of the doctrines of the gospel of Jesus Christ.
Upon the foundation of sola scripture are three massive pillars which frame and uphold the gospel in its most basic formulation—by grace alone, through faith alone, and in Christ alone. And when this foundation and these pillars are in place, the crown can be erected across which is written soli Deo gloria.
After the Reformers, the next giants of faith were the Puritans. These came around the time when Queen Elizabeth began to reign. Few movements in church history have ever been more Bible centered than was the Puritan movement.
I want to consider their view of sola Scriptura under 3 headings:
What are the distinguishing marks out of the Bible itself regarding sola scriptura?
First, it would have to begin with the inspiration of Scripture (2 Timothy 3:16). Every chapter, verse, and book in the canon is breathed out by God. The authors recorded exactly what God wanted them to write. Matthew 4:4 says every word of Scripture comes out of the mouth of God. Hebrews 4:12 says this book is alive. It has the life of God within it. In John 6:63 Jesus says the words I have spoken to you are spirit and life.
Secondly, the inerrancy of Scripture is included in the doctrine of sola scriptura. Since the Bible is God-breathed, and since God is holy, he cannot lie. Thus the God-breathed Bible is totally true. Hebrews 6:18 says it is impossible for God to lie.
Third, the infallibility of Scripture. All that is recorded in Scripture must come to pass. The words of the Lord cannot fail. In Matthew 5:18 Jesus says that until heaven and earth pass away nothing will pass from the Law and the Prophets until all is accomplished. The Scripture cannot be broken (John 10:35).
Fourth, the authority of Scripture. Being infallible, inerrant, and God-breathed, it comes as God’s very words, with sovereign authority. It is the law of the Lord and binding upon every man’s conscience and life.
Fifth, its perspicuity. Sola scriptura means the word of God is clear and understandable, a lucid revelation. In Matthew 19:4 Jesus asks, “Have you not read?” implying that God has so clearly spoken in his word that no man can render the excuse that he did not understand what God was saying. We need the illumination of the Holy Spirit, yes, but any shortcoming in understanding the Bible is not on the part of the Bible but on the part of man.
Sixth, sola scriptura implies the sufficiency of scripture. The word of God is able to accomplish all of God’s purposes here on earth as his word is brought to bear on the men and issues of this world. God’s word will not return to him empty or void. God’s redemptive purposes will be carried out in this world by the word of God which is powerful to convict, convert, conform, console, and correct. It is powerful, more powerful than any other object you and I will ever hold in our hand.
Seventh, sola scriptura implies the immutability of Scripture, that it will never change but always be the same. Psalm 119:160 - “Every one of your righteous ordinances is everlasting.” Right will always be right. Wrong will always be wrong. The way of salvation will forever be the way of salvation. God’s word is unchanging because God is unchanging.
Eighth, sola scriptura also implies the invincibility of Scripture. It is the superior weapon. Sharper than any two-edged sword.
Ninth, and finally, sola scriptura implies the finality of Scripture. That there is no new revelation to be given to man after the close of the canon of Scripture, the faith once and for all delivered to the saints (Jude 3).
All of these truths are inherent in the statement of “sola scriptura.” The ninth point is particularly important to our conference here: the exclusive finality of divine revelation in the written word of God.
The Puritans convened in 1643 to begin writing the Westminster Confession. In 1646 they completed their draft and ratification of the Westminster Confession of Faith. One hundred and twenty-one scholars, theologians, pastors and teachers held over 1100 sessions. They also wrote the Shorter and Longer Catechisms.
Chapter 1 of the Confession begins “Of the Holy Scriptures.” As new challenges had arisen since the Reformation, they had to be more explicit and detailed on this point than ever before. They saw it necessary at the very outset to defend the idea that revelation has ceased and has been captured in full in the Scriptures. They then go on to defend the inspired nature of the Scriptures and how they are now the rule of faith and life. After this they state that the Apocrypha is not part of the Bible, but that only the 66 books of the Old and New Testaments are.
The fourth section of Chapter 1 states that the authority of Scripture ought to be believed and obeyed. In the sixth section they write of its sufficiency. There is no need for appendixes to supplement what has been recorded in the Bible. Here in the sixth section is another cessationist statement. It says that nothing at any time is to be added, whether by new revelations of the Spirit or traditions of men.
In sections 7 and 9 of Chapter 1 they deal with the perspicuity of Scripture—all the things that pertain to salvation are clearly propounded in the Bible. Everything you need to know to be saved and follow the will of God is clearly spelled out in this book. It’s not hard to understand, it’s just hard to swallow. They also go on to say that not only the learned but also the unlearned may attain to a sufficient understanding of them. Section 9 says that Scripture is the best interpreter of Scripture. If something is unclear in one place, another place in Scripture explains it. Finally, in section 10 is a summation of the chapter that states the authority of the word. The Holy Spirit himself speaks in the Scriptures. This is where he has chosen to speak.
This is the commitment of the Puritans in 1646. The church has always been the strongest when it takes this stand on the word of God. If the church takes one step off of sola scriptura, she puts her foot on the slippery slope leading to liberalism, ecumenicalism, agnosticism and eventually atheism. Every church, seminary, denomination that goes astray goes astray at this point. This is essential. We’re not just dogmatic about this; we’re bulldogmatic about it. It is not up for debate.
Whenever God opens the heavens to bless his people, the devil opens his mouth to blast them. At the exact same time as the Westminster Divines were writing the Confession, the Quakers were forming. They claimed to be receiving new revelations and prophecies. They were lead by a man named George Fox. At the heart of their theology was this message, that one can be saved apart from the Scripture, that there is an inner light in all man, and this inner revelation makes salvation for all humanity possible. They called this light within the “indwelling spirit” which they claimed was even in unbelievers.
As they gathered together, the Quakers claimed that they had the Holy Spirit within them. Their worship services had no ordained pastors. They would all sit in a building like we’re sitting here and would be encouraged to meditate, and as you would feel prompted, you were encouraged to stand up and speak your thoughts to others. This commitment to be open and uncautious led them into all kinds of bizarre behaviors and beliefs, including going naked as a sign of judgment to others.
I’m telling you, if you take one step off of sola scriptura you have put your foot on a theological banana peel that will send you down till you hit bottom.
John Owen gave himself to address the fanaticism of the Quakers. In 1659 he responded by writing “A Defense of Sacred Scripture Against the Fanatics.” I want to zone in on chapter 3 “On the Perfection of Scripture”. Here he states his case for the lunacy of these charismatic excesses on the perfection of the written word of God.
He argues that Genesis to Revelation has everything necessary for the believer, that it is perfect as it stands, and that any attempt to add to it is an attack on the perfection of it.
He writes that the Scriptures are the settled, ordinary means of grace, a perfect and unshakable rule for divine worship that leaves no room for any new revelations. If you are seeking any of these charismatic new revelations it is because you have no understanding of the perfections of the word of God.
Owen asks what is the practical good of Scripture if it needs poor mortal man to continue to add to it? He then lists seven consecutive paragraphs in defense:
1662 is a year that will live in infamy in church history. It was in this year, two years after Charles II came back to restore the monarchy in England, that the English parliament passed the Act of Uniformity. This required every preacher in the Church of England to conform to a prescribed set of doctrines and rites or else lose their ministries. And on August 24th, 1662 two thousand Puritan preachers were put out of their pulpits in what has become known as the Great Ejection. Two years later another act was passed by Parliament to prevent these preachers from preaching in private homes and other venues. Soon after they were even barred from coming within five miles of any city. This also meant they were now forbidden from being buried in church cemeteries.
Whenever I go to London I go to Smithfield, and soon after I visit Bunhill fields, where the Puritans were buried outside of the city limits. There you see the tombs of John Owen, John Bunyan, Isaac Watts, Thomas Goodwin, and many other Puritan giants, many of whom have no headstones as they died in anonymity. They were willing to pledge their life and their death on sola scriptura.
May we here tonight have a cessation clause in our personal statement of faith. May we not be given to the errors of this age. May the Word of God regulate all of our lives and ministries and be the only rule of our faith and standard of what is true in this world. May God give us much grace to stand upon the word of God.

A La Carte (10/18) [challies.com - Informing the Reforming] 2013-10-18 06:27
The Death of Death - GLH Publishing continues to release excellent Kindle books; the newest is John Owen’s The Death Of Death In The Death Of Christ and for a limited time it is just $0.99. Also on Kindle is The Dragon’s Tooth by N.D. Wilson, an excellent novel for your young teen or tween ($2.99).
Look & Live (Worship or Die) - “Worship is a looking. It is the gaze of the soul towards greatness….towards some savior. It is the pulsating, throbbing ache of the soul to see & celebrate glory. The ‘wow’ of the heart. And we are always being captivated by something. We must be. The throne of the heart must be filled.”
Pulpits, Pimps and Progress - There’s been a lot of focus in media on the prosperity gospel lately. Here is a lament from a young, Black, Christian male seminary graduate concerned about church perspectives.
Is Public Schooling an Option? - A couple of weeks ago I linked to Al Mohler’s article about public schooling. Jemar Tisby has a thoughtful, insider response.
Recovering a Vision - Make some time to watch this short documentary about Al Mohler.
Serial Trespasser - “Bradley Garrett, a photographer and researcher with a background in anthropology and archeology, has spent the past five years of his life exploring hidden and forgotten parts of cities all over the world.” It makes for an interesting photo gallery.
God’s Pleasure In Me - “I love the truth that salvation is by God’s grace alone, purchased by Christ alone, and gained through faith alone. That message is the unshakable, life-giving foundation of my life. But recently I’ve heard statements about God’s grace that puzzle me. Statements like, You can’t do anything to make God more pleased with you…”
In contending with certain sins there remains no mode of victory but by flight. —C.H. Spurgeon

Strange Fire Conference: A Case for Cessationism [challies.com - Informing the Reforming] 2013-10-17 15:39
Today Tom Pennington spoke at the Strange Fire conference and provided a case for cessationism. He offered seven biblical arguments for the cessation of the miraculous gifts of the Holy Spirit. Here is a summary of his session.
The label “Cessationism” is negative, but the real problem is that it has been easily caricatured as believing that the Spirit has ceased his work. But the fact is that we who are cessationists believe the Holy Spirit has continued his work. Nothing eternal happens in a person apart from the Holy Spirit. Temporal things can happen, but nothing eternal. We only believe the Spirit has ceased in one function: the miraculous gifts, such as tongues, prophecy, and healing.
Charismatics’ chief arguments for believing the gifts have continued are:
Cessationism does not mean that God no longer does anything miraculous. As a pastor I see miracles often. Every time a spiritually dead person comes to faith is a miraculous work of grace. Every time a person is healed solely in answer to the prayers of God’s people totally in contradiction to the medical science predictions, it is a divine miracle. If God so chose, he could allow someone to speak today in a language they didn’t previously know.
Cessationism means the Spirit no longer gives believers miraculous spiritual gifts as a normative Christian experience as it was for the apostles.
Why do we believe this?
Here are 7 biblical arguments for Cessationism:
1) The unique role of miracles. There were only 3 primary periods in which God worked miracles through unique men. The first was with Moses; the second was during the ministries of Elijah and Elisha; the third was with Christ and his apostles.
The primary purpose of miracles has always been to establish the credibility of one who speaks the word of God—not just any teacher, but those who had been given direct words by God. Notice in Exodus 4:15-17 that for Aaron to be Moses’ prophet he could not speak for himself. He could only speak what Moses told him to. This is what it means to be a prophet. But how were the people to know if a man who claimed to be a prophet was in fact speaking God’s own words? Moses brings this dilemma up with God at the beginning of chapter 4, and God answers by giving him signs.
God enabled Moses to perform miracles for one purpose only: to validate his claims to speak for God. This continues to be the purpose of miracles throughout the Old Testament. Only those who spoke authoritatively and infallibly for God were given the power of miracles.
When we come to the New Testament we discover this same pattern. The primary purpose of Jesus’ miracles was to confirm his credentials as God’s final and ultimate messenger (John 5:36; 6:14; 7:31; 10:24-26, 37-38). Jesus’ miracles were not primarily a tool for effective evangelism or about alleviating human suffering. The main reason the Holy Spirit empowered Jesus to perform miracles was to confirm that he was everything he claimed to be and that he spoke the words of God (Acts 2:22). Jesus gave this same power to the apostles, and their miracles served exactly the same purpose (Acts 14:3; cf. Hebrews 2:3-4).
Far more can be shown and said about this, but because we see this pattern throughout Scripture, it is reasonable to conclude that with the death of the apostles and end of their ministry, miracles ceased. Just as they ceased when Moses passed and Elijah and Elisha passed.
2) The end of the gift of apostleship. In two places in the New Testament Paul refers to the apostles as one of the gifts Christ gave his church (1 Corinthians 12:28; Ephesians 4). Although not all spiritual gifts are offices, all offices are gifts to the church.
One of the gifts Christ gave his church was the apostles, but they were a temporary gift. Most agree that there are no more like the original apostles. No one meets the qualifications anymore, which included being an eye-witness of the life of Christ and his resurrection. You also had to be personally appointed by Christ and be able to work miracles (Matthew 10:1-2). According to these three qualifications, there is no one alive today who is an apostle in the sense that the original 12 were. This gift of Christ to the church disappeared after the apostolic age.
This indicates there has been a major change in the gifting of the Spirit between the apostolic age and today.
3) The foundational nature of the New Testament apostles and prophets. The New Testament identifies the apostles and prophets as the foundation of the church (Ephesians 2:20-22). In the context, it is clear that Paul is referring here not to Old Testament prophets but to New Testament prophets. Once the apostles and prophets finished their role in laying the foundation of the church, their gifts were completed.
4) The nature of the New Testament miraculous gifts. If the Spirit was still moving as he was in the first century, then you would expect that the gifts would be of the same type.
Consider the speaking of tongues. At Pentecost, the languages spoken were already existing, understandable languages. The New Testament gift was speaking in a known language and dialect, not an ecstatic language like you see people speaking in today.
Consider also the gift of prophecy. Nowhere does the New Testament distinguish Old Testament prophecy from New Testament prophecy. Just as the Old Testament prophets spoke direct, infallible revelation from God, so did the New Testament prophets. And once it was checked against previous revelation and approved, it was added to the church’s revelation. New Testament prophecy is direct, infallible revelation. Today, however, prophecy is understood differently. Wayne Grudem, who is the most able defender of this position, says modern prophecy should be prefaced with “I think this is what the Spirit is saying.” This is not New Testament prophecy.
There is a disparity between New Testament and modern day healings as well.
5) The testimony of church history. The practice of apostolic gifts declines even during the lifetimes of the apostles. Even in the written books of the New Testament, the miraculous gifts are mentioned less as the date of their writing gets later.
After the New Testament era, we see the miraculous gifts cease. John Chrysostom and Augustine speak of their ceasing. Martin Luther, John Calvin, Jonathan Edwards, Charles Spurgeon, and B. B. Warfield all agree that the gifts ended after the 1st century and had been given only to confirm the message when it first appeared.
This raises a huge problem for our continuationist friends. How do they explain the ceasing of miraculous gifts throughout such long periods of church history?
6) The sufficiency of Scripture. The Spirit speaks only in and through the inspired Word. He doesn’t call and direct his people through subjective messages and modern day bestsellers. His word is external to us and objective. Steve Lawson will have more to say about this.
7) The New Testament governed the miraculous gifts. Whenever the New Testament gifts of tongues was to be practiced, there were specific rules that were to be followed. There was to be order and structure, as well as an interpreter. Paul also lays down rules for prophets and prophecy. Tragically most charismatic practice today clearly disregards these commands. The result is not a work of the spirit but of the flesh.
These are seven biblical arguments for Cessationism. How should you respond?
If you are a cessationist, don’t overreact and downplay the work of the Holy Spirit work in your life and ministry. Hold to your confidence in the all-sufficient Word. We may soon be in a minority, but we stand in the historic position of the church and in the Word. Respond wisely to the different kinds of continuationists. To the charismatics who have bought into the prosperity gospel, confront them with the biblical gospel. Challenge them to test whether they are in the faith. Graciously clarify the biblical argument for why the gifts were given and help them understand.
If you are unconvinced, don’t allow yourself for the sake of peace to ignore the biblical arguments we have raised today. Don’t accept the “open but cautious” position just because it is popular, but test the Scriptures yourself to see what they say.

Strange Fire Conference: John MacArthur Tests the Spirits [challies.com - Informing the Reforming] 2013-10-17 13:51
The second day of the Strange Fire conference began with John MacArthur preaching a message titled “Testing the Spirits.” It was based on 1 John 4: “Beloved, do not believe every spirit, but test the spirits to see whether they are from God…”
There are many places in the New Testament where we are told to test all things and this is critical because Satan and his demons exist and because they operate a kingdom of lies that dominates the world. Satan has been allowed to run loose in this world and he and his agents are disguised as angels of light. We should not be surprised that Satan operates 99% of the time in false religion, in lies and deception. He is not the one behind the corruption in sinful society—the flesh takes care of that. He is behind the false systems of belief that pervade this world.
MacArthur said that many Christians get spiritual warfare all wrong and turned briefly to 2 Corinthians 10:3ff where we see that the weapons of our warfare are not human and that we cannot rely on anything concocted by man. Our weapons must be divinely powerful. Why? Because we must be engaged in the destruction of fortresses. The picture here is that human weapons are no match for a huge and impregnable fortress. We are assaulting formidable edifices and cannot use pea-shooters. These fortresses are speculations, ideas, psychologies, and religions. Spiritual warfare is not about running off demons, but battling for the mind.
Why? Because the world is imprisoned in belief systems and worldly people are fortified in them. These belief systems become their prisons and end up being their tombs. The architect of it all is Satan, the arch-deceiver. These fortresses are further defined as “every lofty thing raised up against the knowledge of God.” This refers to every great insight or noble idea, everything raised up as an ideology against the knowledge of God.
What is our responsibility as Christians? It is to smash these ideologies, to crush these fortifications, and to take every thought captive to the obedience of Christ. Once again, we need to remember that we are engaged in a battle for how people think.
All throughout the Old and New Testaments we are warned about false prophets. We are warned to ensure we continue to be faithful to the truth. A key component of fighting this war is testing the spirits. We cannot go blithely along accepting whatever people aver or avow. We need to test and 1 John 4 gives us the tools to do that.
When the Great Awakening broke out, there was much debate about what was and what was not a true work of the Spirit. Jonathan Edwards went to 1 John 4 and MacArthur closely followed Edwards’ The Distinguishing Marks of a Work of the Spirit of God. (Note: Time did not permit him to get too far into this; the Strange Fire book contains a much more detailed examination.)
We are all responsible to assess anything and everything that is claimed to be a work of the Holy Spirit. These are timeless tests for all movements, all preaching, and all preachers. What is truly of the Holy Spirit will conform to these marks.
The context for this passage is the work of the Spirit (see 3:24). While the working of the Holy Spirit is invisible, the manifestations of his work are visible. We know Christ abides in us because the Spirit he has given is manifested in us. What is the Spirit doing in us? MacArthur provided a long list. The Spirit creates a desire for repentance, a hatred of sin, a belief in the gospel, a love for Christ, a desire to be a slave of Christ, a delight in Scripture, a longing for obedience, joy in trials, love of other believers, desire for fellowship, illumination of Scripture, a heart of praise, worship as a way of life, increasing Christ-likeness and much more besides.
But there are other spirits and it is noteworthy that in a sudden and unexpected shift from the glories of 3:24, the Holy Spirit brings words to John’s mind that move from the glorious reality of the true work of the Spirit to the deadly dangers of unholy spirits that are not from God. But we should not be surprised because we have always been warned that Satanic deception is with us. It is always at work. God has always warned his people and called them to vigilance and discernment. This war against the truth rages all the time.
It is a telling irony to MacArthur that if you criticize the charismatic movement and endeavor to be vigilant, and if you endeavor to hold them to Scripture while exposing error, they will condemn you as the sinner. Suddenly you are the one who is standing in the way of Christ’s prayer for unity. In order for this movement to succeed, they have to turn discernment into a transgression against Christ and his high priestly prayer. For the charismatic movement to survive, it must attack truth warriors and turn them into enemies of the Holy Spirit. The reason is simple: if sound doctrine rules, charismatic theology does not survive.
He turned back to 1 John 4 where we are told, Beloved, do not believe every spirit (which means every person). Why? Because many false prophets have gone out into the world. The simple fact that the charismatic movement does not want examination ought to convince you of its error. If this was a true work of the Spirit, they would be inviting all the scrutiny they could get. They would want the affirmation and authentication.
So what are the tests that Edwards drew out of 1 John 4?
Test One: The true work of the Holy Spirit exalts the Lord Jesus Christ (see verse 2). The first test is Christological. Any Spirit that confesses Christ is from God; any spirit that does not confess Christ is not from God. It’s that simple. False religions and heretical cults all have an aberrant Christology.
His major critique of the charismatic movement is that it focuses undue attention on the Holy Spirit and does so at the expense of Christ. Any true preacher will be Christ-dominated and present him in an accurate and exalting way. It is a matter of sound theology and also a matter of preeminence. Where you see any deficiency in the nature and preeminence of Christ, this is not the work of the Holy Spirit. The Holy Spirit’s ministry is always to point you to Jesus Christ. Anyone who pollutes the gospel or distracts from the Son to the Spirit is not operating in the Spirit.
The devil would never want men to have more honorable thoughts of Christ and for that reason loves to draw attention away from Christ to a false image of the Holy Spirit. And all the while he pretends to draw attention to Jesus. A true work of the Spirit exalts the true Christ. If the charismatic movement was a movement of the Spirit, it would be Christ-dominated and everyone in the movement would be bowing the knee to the true Christ in belief of the true gospel.
According to leading charismatics, a distinctiveness of the charismatic movement is the preeminence of the Holy Spirit. They have a passion to experience the Spirit’s presence and power. But if the Spirit is the person sought, his work has been rejected. In this movement Christ is obscured, Scripture is depreciated, and a preoccupation with experience is elevated.
The charismatic movement fails this test of exalting Christ above all. MacArthur said, Show me a person obsessed with the Holy Spirit and I’ll show you a person not filled by the Spirit. Show me a person obsessed with Jesus Christ and I’ll show you a Spirit-filled person.
Through the rest of the time allotted to him, MacArthur displayed and illustrated some of the worst of the charismatic movement, largely focusing on successful and influential leaders like Kenneth Copeland and Creflo Dollar. All the while he meant to show that so much of the silliness, the heresy and the misrepresentation of the gospel stems from diminishing Jesus Christ in favor of the Holy Spirit.
We know that the Spirit would never devalue the gospel, so wherever the gospel is devalued, we know the Spirit is not in it. He highlighted a few gospel misrepresentations: the synergy between charismatics and Roman Catholic charismatics (If a movement can embrace Catholicism, it is not a movement of the Holy Spirit), Oneness Pentecostalism (if you don’t have the true God, you don’t have the true Spirit) and the health and wealth gospel (the prosperity gospel has no interest in the biblical gospel). If we were to add up the people connected to just these three heretical groups, we would have a majority within the charismatic movement.
Unfortunately he ran out of time long before he could give fair attention to Edwards’ other tests. However, he mentioned that his book (which every conference participant will receive) contains all of this in much greater detail.
And he concluded like this: When it comes to charismatic theology and manifestations of the Spirit’s work, we do not need to speculate; we have the tests and simply need to apply them.

Strange Fire Conference: First Day Reflections [challies.com - Informing the Reforming] 2013-10-17 11:48
The first day of the Strange Fire conference has come and gone and the second is just about to begin. To this point it has certainly lived up to its billing as an event that will deal frankly with concerns related to the charismatic movement. I want to offer just a couple of brief reflections.
The format has been very deliberate and rather interesting. John MacArthur began with a series of direct statements about charismatic theology and was followed by Joni Eareckson Tada who provided a testimony of how God has chosen not to heal her paralysis and chronic pain. R.C. Sproul brought a theological perspective to Pentecost, Steve Lawson brought a historical perspective from John Calvin, and Conrad Mbewe showed how charismatic theology in its worst form has come to dominate African Christianity.
Here is a one-sentence summary of each of the addresses so far:
Judging by comments and by social media buzz, the event is being closely followed by many who hold to charismatic theology, and especially those who also hold to Reformed theology. Some are expressing sorrow at what they see as harsh and unfair treatment while others are expressing either patience or gratitude. Many are confused and are hoping for clearer definitions and positive affirmations that will better express and defend the cessationist position.
Until the day of the event, and really until the end of MacArthur’s opening address, I was unsure of whether or not I would give a lot of attention to the event. But I am glad I chose to blog about it as it really does seem to be making a big splash in the Evangelical world and especially among the Reformed crowd that tends to read this site. Like you, I am very interested to know what will come today and tomorrow.
What remains to be seen, and what may take quite a long time to see, is whether this event will call Christians to work to find greater agreement on the issue of the miraculous gifts, or whether it will polarize the camps even further. It is fast becoming my prayer that one way or another the Lord will see fit to use this event to bring greater maturity and greater unity to his church.

The Christian Conscience [challies.com - Informing the Reforming] 2013-10-17 08:17
Over the past few weeks Dr. Joel Beeke and I have been teaming up to work our way through a portion of his massive new work A Puritan Theology. We have not been reading the whole book, but just the final eight chapters which deal with practical theology, the “so what?” of systematic theology.
This week we read chapter 56 which discusses the Puritans and conscience. I asked Dr. Beeke a few questions related to the Puritans and the way they understood matters of conscience.
TC: In order to ensure we are all on the same page, can you define conscience? What exactly is it the Puritans were talking about when they discussed matters of conscience?
JB: The conscience is an echo in the human mind of the verdict of the righteous Judge. William Perkins said that “conscience is a part of the understanding” that sets itself either for or against their actions. William Ames, a student of Perkins, wrote that conscience is “a man’s judgment of himself, according to the judgment of God of him.” Regardless of what we love with our affections or choose with our will, there is a part of our understanding that judges us and makes gives us a sense of moral approval or guilt according to our understanding of right and wrong. So when the Puritans considered cases of conscience, they were discussing questions about how to know what is pleasing to God in specific situations, and most importantly how to know that the divine Judge accepts you as righteous in His sight.
TC: What would the Puritans identify as the function of conscience? Why do we need it and what does it do for us?
JB: Conscience impresses a man’s mind with the moral authority of God, and as a result produces a sense of anxiety and misery, or peace and joy, that anticipates eternity. Ames said that conscience binds a man with such authority that no created thing can release him from it Though our conscience may be misinformed, still it speaks with a divine authority that we may disobey but we find difficult to ignore. It reminds us that God sees all we do, and that He is either delighted or angry with our persons, and pleased or displeased with our deeds.
Much Puritan literature aimed to direct people to find peace of conscience through the blood of Christ, and to walk in good conscience day by day. Richard Rogers said that his purpose in his Seven Treatises of spiritual guidance was to show a person how to live such that “he may find a very sweet and effectual [powerful] taste of eternal happiness, even here.” Richard Sibbes said that a good conscience is “a continual feast,” because knowing that God is pleased with us, has forgiven our sins, and delights in our obedience, enables us to suffer and even to die with comfort, freedom, and joy.
TC: What would the Puritans want us to know about the effect of the fall into sin on man’s conscience?
JB: The fall of man brought us under the condemning wrath of God and the enslaving darkness of sin. The first disturbs and terrifies the conscience insofar as it senses the coming judgment. The latter disorders and confuses the conscience.
Perkins taught that though a “remnant of God’s image” persists in man’s mind through “certain notions concerning good and evil,” mankind has fallen into much ignorance of the truth and inability to understand spiritual realities (1 Cor. 2:14), futility in not distinguishing truth from falsehood (Eph. 4:7; Prov. 14:12), and natural tendency to follow evil and lies (Jer. 4:22). This distorts the conscience, though it still retains a degree of its power to rebuke and restrain sin (Rom. 2:15). Fallen conscience tends to excuse inward wickedness if it is covered in outward worship (Mark 10:19–20). It also tends to falsely accuse a person when he fails to follow the traditions and doctrines of mere men (Col. 2:21–22). Sometimes conscience may accuse and terrify a person for his sins (Acts 24:26), and yet consciences may be seared to numbness by habits of sinning (Eph. 4:19; 1 Tim 4:2).
TC: Where might the Puritans warn us about our use or misuse of conscience?
JB: The Puritans warned against subjecting conscience to any ultimate authority besides the Bible. They particularly emphasized liberty of conscience in matters of religion. The Westminster divines wrote, “God alone is Lord of the conscience, and hath left it free from the doctrines and commandments of men, which are in any thing contrary to His Word; or beside it, if matters of faith and worship.” Similarly, the Particular Baptists wrote, “The Holy Scripture is the only sufficient, certain, and infallible rule of all saving knowledge, faith, and obedience.”
The Puritans also warned against resisting one’s conscience when it speaks according to the Word. Ames taught the unconverted to seriously consider the law so that it may convict him of sin, show him he cannot save himself, and bring him to grief, fear, and confession of specific sins. He must also renounce his own righteousness and fix his mind upon the righteousness of Christ crucified as presented in the promises of the gospel.
Christians too must not resist conscience. If a Christian finds his conscience accusing him, Ames counseled him to: first, feel the burden of sin (Matt. 11:28–29); second, detest all sin (Rom. 7:15); third, be careful not to fulfill his sinful lusts (Gal. 5:16); fourth, work to put those lusts to death (Rom. 8:13); fifth, to consider God’s promises, flee to Christ, and cling to Him more and more (Rom. 7:25; Phil. 3:9); and sixth, get rid of gross and heinous sins that shake their consciences and call into question their very salvation (Isa. 1:16–18).
TC: What can a Christian do to repair his conscience or to help his conscience overcome the effects of the fall?
JB: The restoration of the conscience is part of the process of sanctification that begins with regeneration and does not end until we enter glory. It is a work of God’s grace that we must seek in prayer. The most significant means is to place ourselves under the sound and searching preaching of both the law and the gospel. As Sibbes said, the steps to a good conscience are first to be troubled by our sins, second to find peace by trusting in Christ, and third to resolve to please God in all things. With these three elements active in our lives, we are positioned to grow more in a good conscience as we live by faith for God’s pleasure. The most important attitude is honesty and humility before God, for conscience always confronts us with the truth that God is Lord. For more details on restoring the conscience, see A Puritan Theology (pp. 919–25).
If you are reading along with us, be sure to read Chapter 57 (“Puritan Casuistry”) by next Thursday. Then simply check in here to see what Dr. Beeke has to say about it.
The purpose of this project is to read classics together. Please feel free to leave a comment below or to provide a link to your own blog if you have discussed this week’s chapter there.

Strange Fire Conference: Conrad Mbewe [challies.com - Informing the Reforming] 2013-10-17 06:40
Conrab Mbewe is a man who wears many hats and who fulfills many different responsibilities, but above all else he is a preacher of God’s Word. MacArthur introduced him by explaining that he wished to have Mbewe at the event because the charismatic movement has done devastating damage in Africa and he wanted an insider’s perspective. Mbewe titled his message “The African Import of Charismatic Chaos.” Here are some brief notes.
Mbewe decided to provide a brief overview of the charismatic movement in Africa. It is a movement he has observed for over thirty years and one that is of great concern to him. This is not something he has learned about by reading books, but something he comes across literally every day. He warned that some of what he would say would be somewhat foreign to a Western mindset, but he felt it necessary to speak from his African background.
He went to John 17:17 and said the charismatic chaos we see would never have been the case if this verse had been taken seriously. This verse comes near the end of Christ’s ministry, on the eve of his crucifixion. He is seeking to convince his disciples concerning how they ought to live in his absence, and his great desire is for God to be glorified. In the time between Jesus’ ascension and his return, God’s Word is to remain serving and sanctifying his people. This is what Jesus desires, yet the African charismatic movement has come about because of a failure to hold to the centrality and sufficiency of Scripture.
The charismatic movement has flooded the African continent south of the Sahara Desert where it has become the most visible form of “Evangelicalism.” The phrase “born again” is equated to that form of Christianity. Its spread has largely come through the use of crusades, radio, television and free literature. Most of this literature has been shipped from the United States and it contains the kind of heresy that has become a common diet in the health and wealth movement.
Invariably, this has been riding on the back of the old time conservative Pentecostalism which found its way into much of English-speaking Africa in the second half of the last century. That opened the doors slightly but they have now been blown wide open. Many people ask the question, “Why in this short time has there been such an acceptance, a multiplication of churches that can be described as charismatic?” The answer is that this form of Christianity has appealed to the African worldview in terms of its understanding of the spiritual world. Almost invariably the messages you hear are like this: Come and receive your deliverance, your healing, and your breakthrough. As an African, there is a whole world in his mind that this invariably floods into. The word “breakthrough” is really saying to the common African man that if you are struggling in your marriage or struggling to conceive or struggling to maintain a job (and so on), it is because between you and God there are other layers that need to be dealt with. One of those layers is that of angels and demons and the other is that of your ancestral spirits. Until those layers are broken through, you will not get what you want. This is what the charismatic movement has taken on when dressed in African attire. The language that has already been there for centuries in Africa is given a thin veneer of Bible verses. You can understand, then, that if men and women are running in throngs to the witch doctor, they will rush in throngs to these so-called churches because it boasts the same power they are looking for.
What is happening today has gone a number of steps further than where the charismatic movement began. This has happened because the Word of God is no longer playing the role of governing our thinking and our practice. His concern is that when a lot of people look at the full to overflowing churches in Africa and the apparent excitement in the church, they cross back to the USA and say, “There is revival there.” But the fact is, they are seeing bad news, not good news. It is bad news primarily because of John 17:17. It is bad news because of the absence of serious interaction with the Word of God within the movement.
Thirty years ago you could attend a Pentecostal church in Africa and a pastor would stand in the pulpit and give you some kind of biblical exposition. You might disagree, but there would be some attempt to teach what the Bible is saying. There would be a mid-week Bible study. That is now almost completely absent. You cannot have spiritual life when the Bible is closed! The gospel has been lost and what has replaced it is twenty minutes of motivational speaking followed by an opportunity to bring your problems to Jesus so Jesus can help you get over your problems. Unfortunately, this is drawing people so that droves of them are responding to this message. They want their problems to be handled. The result is that churches are full of goats, not sheep.
Where the word of God is closed, the Gospel has been lost and the way of life becomes sinful and self-centered. This leads to a loss of true worship. Mbewe references the evening worship saying that Pentecostals in Africa have no real interest in singing the kinds of songs that were sung that evening at the conference. Rather, people are singing to danceable tunes. They repeat little phrases over and over again. You can say little phrases that are biblical about the glory of God, but that does not make it worship.
They proclaim that the extraordinary revelatory gifts are still operational today, but the moment you open that door a little bit, where do you stop? There are different levels to the practice, but so many are taking advantage of it for selfish sexual or financial gain. How do you examine your life in comparison to others who have opened their life to these gifts and have taken them to their logical conclusion, those who are successful in areas where you have not been? Mbewe followed this by using himself as an example for taking Pentecostalism to its logical conclusion. He proposes, “What’s to stop someone like me from coming up with irrational ideas because I’ve been empowered to do so?” He has counseled many, many people who are caught in these scandals—sexual scandals about spiritual husbands and wives, where a messenger from God, a pastor, steps in to be sexual partner with someone because of the authority that they have from God. These people keep God’s Word closed.
He concluded by emphasizing his main point about the centrality and sufficiency of God’s Word. God’s Word must be taught and applied. He then shared two reasons why we should be concerned about these issues in Africa. First, Africa’s current population is over a billion people. God is concerned about these people and, therefore, we should be concerned with these people. Second, Africa is strategically placed to be the next major force in world missions and it will be an utter disaster if this is what they export to the rest of the world. Everyone who has the cause of Christ at the center of his or her heart should be concerned about this.
His final remarks expresed his relief to see the Reformed movement growing on the African continent, though it is still in its infancy there. He exhorted us all: We have got to pray and get back to the Bible! Today we are not saying enough that this book is sufficient. It is sufficient!

A La Carte (10/17) [challies.com - Informing the Reforming] 2013-10-17 05:57
Kindle users may want to look at a couple of deals: One Race, One Blood by Charles Ware and Ken Ham ($4.99); The Pursuit of God by A.W. Tozer ($3.91). Meanwhile, Logos users will want to grab this free download: Brothers, We Are Not Professionals.
Quick Tips on Spiritual Growth - Erik Raymond offers some “quick tips on spiritual growth.” But don’t be fooled—there’s nothing quick and easy in the Christian life!
Twenty Years and Counting - Be sure to read this amazing account of Al Mohler’s time at Southern Seminary. Few people know or remember the kind of hostility he faced when he first began there. He also talks about the systematic theology he hopes to write in the future.
Packer’s Systematic Theology - Michael writes about J.I. Packer’s long-awaited Systematic Theology. He and Packer are looking for prayer that Packer will be able to complete it.
Broken Vows - Aaron Armstrong reviews a recent Cruciform Press title, Broken Vows: Divorce and the Goodness of God. And while on the subject of divorce, TIME has a few points about why second marriages are often more difficult than first marriages.
The Russia Left Behind - “On the jarring, 12-hour drive from St. Petersburg to Moscow, another Russia comes into view, one where people struggle with problems that belong to past centuries.” It makes a very interesting article.
The church is a school for sinners, not a museum for saints. —Anthony Thiselton

Strange Fire Conference: Steve Lawson [challies.com - Informing the Reforming] 2013-10-16 21:58
John MacArthur opened the conference with broad statements about the purpose of the conference and what he perceives as the main challenges of the charismatic movement. Joni Eareckson Tada offered her unique testimony and this was followed by R.C. Sproul’s theological perspective. And now added to the mix is Steve Lawson and his perspective from church history.
With the recent resurgence of Calvinism there has been a strange merging of historic, biblical Calvinism with charismatic experiences and worship styles. It has pulled in an entire generation of young, restless, reformed people who believe in miracles, healings, words of knowledge, prophecies, tongues, and so on. They see no reason in the New Testament for why these gifts of the Spirit have ended since the first century.
This merging has gone virtually unaddressed within the reformed community. I believe there is no one better to address these charismatic Calvinists than Calvin himself.
Calvin faced a charismatic crisis of his own in his own day. I want to look at how he addressed them. As the leading reformer in that day, whatever faced the church faced John Calvin. He had the dominant voice and people looked to him to address issues.
The Anabaptists were a collection of subgroups which had elements of an inner word and inner witness of the Holy Spirit. They began to seek ecstatic visions and prophetic manifestations and miracles, etc.
Then there were the Libertines who were one of the subgroups under the Anabaptists. They were antinomians. They abused Christian liberty and proved themselves to be, most likely, unconverted. Calvin called them a sect one hundred times more dangerous than the Roman Catholic church itself. They were lead by fleshly impulses and believed the Holy Spirit was adding new revelation to the Bible. They set aside the Scripture and wanted to follow the inner impulses that they thought were the Spirit. They lived in open licentiousness. They wanted an easy moral path without having to fight sin or temptation.
These were the things Calvin faced in his day. So the charismatic chaos we see now, in our day, is nothing new. It was prevalent in Calvin’s day, as it has been in other eras as well. So Calvin was not silent about it.
Many of today’s reformed leaders who are open to charismatic teaching and experiences would do well to take heed to what Calvin says about it.
In Calvin’s commentary on Matthew 10:1, he states that the office of apostleship was a temporary office. The apostles were the foundation of the church, and you only lay a foundation only once. They were given miracles to authenticate their authority as messengers of the revelation of God in Christ.
In the 1536 edition of his Institutes—his first edition, at the beginning of his ministry—Calvin wrote that miracles were given to attest to the truthfulness and uniqueness of the revelation of the apostles. And once that revelation was attested to and codified in the New Testament, the miracles ceased. They were no longer necessary. What is necessary is the preaching of the now-written word. In his commentary of Mark 16:17 he indicates this same understanding.
Calvin would say to reformed leaders today who are dabbling in charismatic teaching and practice that they are playing with Pandora’s box. They are opening a door Satan easily uses to lead people astray.
In his commentary on Acts 14:3 he says the true use of miracles was to give witness to the gospel. The written word of God and the Spirit of God can never be separated. Whatever the Spirit is doing in the world is always done in connection with what has been written in the word of God. The Holy Spirit who is the author and teacher of the word of God works in perfect partnership with the very word he has authored. What God confirmed in the book of Acts is sufficient for all people at all times and in all places. The gospel does not need to be re-validated.
Concerning tongues, Calvin states clearly in his commentary on Acts 10:44 that he believes the gift of tongues ceased in the first century. In his commentary on Isaiah 30:1, he states that the word and the Spirit of God are connected, “in opposition to the fanatics, who aim at oracles and hidden revelations without the word.”
In his commentary on Acts 21:9 Calvin states his understanding of first century prophecy. He says it should last for but a short time, “lest the faithful should always wait for some farther thing, or lest that curious wits might have occasion given to seek or invent some new thing every now and then.” He states that God took away new revelation in order to testify that the end of revelation was present in Christ. The fullness of what God wants us to know has now been given to us. The faith once for all delivered to the saints.
In his commentary on Romans 12:6 he states what he understands prophecy to be after the first century: the understanding and clear communication of what has already been revealed. He says the same in his commentary of Hebrews 1:1-2. The word God gave in Christ was the final conclusion. Believing in further revelation implies that the revelation in Christ wasn’t enough.
Institutes 1.9.3 - Word and Spirit belong inseparably together. If you take anything away from Calvin, take this. “For by a kind of mutual bond the Lord has joined together the certainty of his Word and of his Spirit so that the perfect religion of the Word may abide in our minds when the Spirit, who causes us to contemplate God’s face, shines; and that we may in turn embrace the Spirit with no fear of being deceived when we recognize him in his own image, namely, in the Word.”
Institutes 2.15.2 - “This, however, remains certain: the perfect doctrine he has brought has made an end to all prophecies. All those, then, who, not content with the gospel, patch it with something extraneous to it, detract from Christ’s authority.”
The point has been established. I refer you to his treatise against the Anabaptists and his treatise against the Libertines. Why couldn’t he leave the Libertines or Anabaptists alone? Calvin responded, “Even a dog barks when his master is assaulted.” He had to say something to defend the truth of Christ and the welfare of the church.
His critique of the charismatic abuses of his day is that they are foolish, vain, and false. They substitute truth for delusions. They are silly. They separate the mutual bond of the Spirit and the Word. They arouse God’s wrath and lead people to follow their own imaginations.
A charismatic Calvinist is, to Calvin, an oxymoron. It can’t exist.
I want to end this in a very simple way. What would Calvin say to this present generation? The answer is what he said to his own generation:
1) The exclusivity of biblical authority. There is only one stream of revelation or there are two streams. Catholics wanted two streams on one side, the charismatics wanted two streams on the other side. And Calvin said No, there is only one stream of revelation after the first century, the written word of God. Sola Scriptura.
2) The priority of biblical preaching. Calvin understood that when you look to more than one stream of revelation, you diminish the pulpit. Having one stream—the word of God—necessitates biblical preaching. Having other streams only downplays and marginalizes biblical preaching.
3) The unity of Spirit and Word. Calvin was convinced that only one stream of revelation joins together the Spirit and the Word in their tightest bond. Two streams drives a wedge between the two. The Spirit is at work only where the written word of God is being preached, shared, taught, read, etc.
Calvin towers over church history as the most substantial theologian who has been given to the church. We would do well to hear from him.
I will let him have the final word. “Let the pastors boldly dare all things by the word of God… Let them constrain all the power, glory, and excellence of the world to give place to and to obey the divine majesty of this word. Let them enjoin everyone by it, from the highest to the lowest. Let them edify the body of Christ. Let them devastate Satan’s reign. Let them pasture the sheep, kill the wolves, instruct and exhort the rebellious. Let them bind and loose thunder and lightning, if necessary, but let them do all according to the word of God” (Sermons on the Epistle to the Ephesians).
If we are to see a new reformation in this day, continue to expand its borders and have greater influence in the church and the world, we must be committed exclusively to the written word of God.

Strange Fire Conference: R.C. Sproul [challies.com - Informing the Reforming] 2013-10-16 21:46
For the third session at Strange Fire, John MacArthur introduced his good friend R.C. Sproul. Because of issues with his health, Sproul was unable to travel to California, so instead he sent along a video message. And his task was to speak about Pentecost.
He began by saying, “I want to look specifically today at the redemptive-historical significance of Pentecost.” We’re aware that the modern Pentecostal movement began at Azusa Street and that it occurred outside of the mainline denotations until the middle of the 20th Century. Then it moved into Catholic, Lutheran, Methodist, Anglican, etc. circles. Initially when it came into these various denominations there were several attempts to assimilate the theology into their creedal foundations. At the same time, Pentecostals were gathering their beliefs into a creed, which became Neo-Pentecostal theology.
One of the most significant aspects of this theology is the idea that it is normal or even normative for people to have the baptism of the Holy Spirit after their conversion. It is admitted that some people can have conversion or regeneration simultaneously with their baptism by the Holy Spirit, but in the main there is a time difference between original conversion and the baptism of the Holy Spirit. It is this particular point I want to address today.
The fundamental weakness of Neo-Pentecostal theology is that it understands the original Pentecost differently than the apostles, and that it considers this Pentecost too lowly. The significance of the baptism of the Holy Spirit has to do principally with the Holy Spirit empowering Christians for ministry. When Jesus promised the Holy Spirit he was promising power and strength.
In the Old Testament a person could only be a believer by being born again of the Holy Spirit. But the difference between the Old Testament and the New Testament with respect to Pentecost is that in the Old Testament the Spirit was only given by God selectively to isolated individuals, such as the prophets or the judges when they needed strength for the particular task.
The most Spirit-endowed person in the Old Testament was Moses. His miracles and leadership were worked out through the extraordinary endowment of the Holy Spirit. He is called the mediator of the old covenant anticipating the mediator of the new covenant who was even more heavily endowed with the Holy Spirit. There came the point in Moses’ ministry when he could hardly bear the burden of leadership any longer. Numbers 11:24ff tells us that the Lord took of the Spirit that was upon Moses and placed the same upon the 70 elders, and when this happened they prophesied. Joshua saw this and asked Moses to forbid them. Moses’ answer is important to our understanding of Pentecost. He replies, “Are you jealous for my sake? Would to God that everybody would get the Spirit!” This wish of Moses’ later became a prophecy in the mouth of Joel (Joel 2:28-29). So what was first a prayer or a wish of Moses became a prophecy of Joel’s for the future.
Fast forward to Acts 2:1ff. Those who were observing the events thought that the speaking in tongues was a result of drunkenness. But Peter responds that this is rather the fulfillment of the prophecy Joel had made. The apostolic interpretation of the day of Pentecost was that it was a fulfillment of Joel’s prophecy. Those who were gathered on that occasion were gathered for a Jewish feast—they were Jewish believers. And notice that all present received this endowment from God. There were no haves and have nots. The Spirit fell upon people who had been believers and now received the Holy Spirit. So you can understand how our Pentecostal friends would see the baptism of the Holy Spirit as a secondary experience for Christians. But because this was a unique redemptive historical event, it was not intended to become a model for how every Christian should experience the Holy Spirit.
The book of Acts follows the Great Commission of Jesus—showing the gospel moving from Jerusalem to Judea to Samaria and to the ends of the earth. There were four distinct people groups with which the book of Acts is concerned: the Jews, the God-fearers, the Samaritans, and the Gentiles. The God-fearers were for the most part Hellenistic Greeks who had converted to Judaism. They believed in Yahweh and embraced the teaching of the Jewish community, but they were not fully accepted because they had not submitted themselves to circumcision. When the new covenant comes along, the question becomes, “Where do these people fit?” As the book of Acts progresses, we see then not one Pentecost experience but four. We see four outpourings of God’s Spirit upon certain people.
In Acts 8:14-17 we have the record of what happened among the Samaritans. There is a second Pentecost among the Samaritan believers when Peter and John lay hands on them. In Acts 10:44-48 the Spirit falls on the God-fearers, which Peter recounts in 11:13-18. This is Pentecost number three. Just as in the case of the first and second Pentecosts, all of those present received the Holy Spirit. In Acts 19:1-7 the Gentiles in Ephesus receive the Holy Spirit and are empowered for ministry.
So you have four separate Pentecosts, one for each people group in Acts. When Paul was dealing with the Corinthian church, he wrote in 1 Corinthians 12:12-14 that by one Spirit we were all baptized into one body. Here he speaks of the universality of the Sprit’s empowering of every believer. That’s the significance of Pentecost.
In Ephesians 2:11-19 Paul again addresses this issues that threatened to divide the 1st century church, the issue of what role the Gentiles have in the body of Christ. Paul’s “mystery” in Ephesians and Colossians is that Christ has folded Gentiles into his body and indwells them. “Through Christ we both have access through one Spirit to the Father.” This is a Trinitarian work.
My concern with Charismatic friends is that they have a low view of Pentecost. They don’t see it as a signal of the outpouring of God on all Christians. They believe all Christians can have it and should have it, but they miss the point that the pouring of the Spirit at Pentecost means that all Christians already have the Spirit and have been empowered by him, and that they don’t need to be baptized by the Spirit again.

Strange Fire Conference: Joni Eareckson Tada [challies.com - Informing the Reforming] 2013-10-16 17:36
John MacArthur opened the Strange Fire conference, and then, for the second session introduced Joni Eareckson Tada as a friend and former member of his church. She was at the conference to share her testimony of living as a quadriplegic who has prayed for, but not received, a miraculous healing. As MacArthur said in his closing comments, if anyone has the faith to be healed, it must be her. In a sweet and spontaneous moment, Joni called MacArthur to the stage and, hand-in-hand, the two sang a couple of stanzas of “O Worship the King” together. I have been to a lot of different conferences, but that will now rank as one of my all-time favorite moments.
Joni began by reading John 5, the story of Jesus at the pool of Bethesda healing a man who had been paralyzed for thirty-eight years. She followed it with her story of going to see Kathryn Kuhlman, hoping that she would be healed and rise from her wheelchair. But Kuhlman did not heal her, and Joni wondered who this God was who would deny her what she was sure she needed. A bitter spirit began to take hold of her. If she couldn’t be healed, she wanted to be left alone in her despair.
When she did turn to the Bible, she had a special interest in healing, but soon saw that physical healing was not Jesus’ main interest; he was far more concerned with spiritual health. She realized then that her interest in Jesus had been more for what he might do to heal her body than for how she might serve him. That is when she began searching for a deeper healing and once she understood that healing, the Lord taught her that her disability was a means through which God was causing her to grow in holiness.
She went on to speak of the chronic pain that lasted for many years and the stage three cancer that followed it and expressed how she has learned to be grateful for the suffering because of the way it keeps her longing for Christ. The suffering that results from sin in the world, God now uses to get rid of sin. There is nothing sweeter than knowing the joy of the Lord Jesus in the midst of suffering and all the while she holds on to the hope and the confidence, that in heaven, the big deal won’t be getting a new body that works, but a glorified heart that no longer twists truth, becomes anxious, manipulates others, and all these other manifestations of sin.
Even today she often has well-meaning charismatics who come up to her and pray for her healing. Though she never says no, she does always ask them to pray for specific things and then highlights character issues. Will you pray for my bad attitude? Will you pray for my grumbling? She means to show them that she is far more concerned with indwelling, remaining sin than chronic pain and legs that do not work.
She went on to describe a trip to Jerusalem and going to the very place where Jesus had healed that paralyzed man so many years ago. And there, in a moment alone, she found herself praying to God to thank him for not healing her, because a “no” answer to her requests for physical healing had purged so much sin, selfishness, and bitterness. That “no” answer left her depending more on God’s grace, has given her greater compassion for others, has reduced complaining, has increased her faith, has given her greater hope of heaven, and has caused her to love the Lord so much more. She sees the joy of sharing in his suffering and would not trade it for any amount of walking.
And she closed with this question: When you see yourself today, do you see yourself waiting at the side of the pool of Bethesda? Are you wondering why God hasn’t removed the disappointment and given healing when you’ve asked for it. God may remove your suffering, but if not, he will use it to destroy sin. This is the deepest healing, and you do not need to break your neck to receive it.
And then, in classic Joni style, she led everyone in prayerfully singing the hymn “Have Thine Own Way, Lord.”

Strange Fire Conference: John MacArthur's Opening Address [challies.com - Informing the Reforming] 2013-10-16 15:08
I am always intrigued by current trends in the Evangelical world, and especially in this Reformed corner of the Evangelical world. When something comes along that seems as if it will make a significant impact, I like to take note. For that reason I have been tracking with what John MacArthur is attempting through his Strange Fire conference and book. That conference kicked off just a short time ago and I listened with interest (via livestream) to the opening keynote because it was here that MacArthur would give his rationale for the event and it was here that he would set its tone.
Before I share my notes, let me say just one thing that stood out to me as the conference began. It is inevitable that at some point John MacArthur will be the subject of a biography (beyond the existing biography written by Iain Murray). Today he is beginning something that will, I think, appear in that biography. We will know better as the conference unfolds what impact it will make in his life and ministry and in the wider Christian world.
There are 4,000 people in attendance at the Strange Fire conference and many thousands more who are watching the live-stream in English, Spanish, German, Portuguese, Arabic, Italian, French, Russian and Mandarin. Here is what they heard in the opening address.
As we address the contemporary Charismatic movement, we are addressing a subject that has been a concern of MacArthur’s for many years and decades. Back in the early days of his ministry he saw the early beginnings of the movement and was deeply concerned. He has addressed it many times since then, first in a series 40 years ago, and later in the book Charismatic Chaos.
When people ask MacArthur for his view on the biggest issue in the church, he always says it is the lack of discernment since, sadly, a great number of those who profess Christianity are lacking in discernment. The purpose of this conference is to be like the Bereans by looking at the work of the Holy Spirit through the lens of Scripture. He hopes to address it lovingly and compassionately, but in a straightforward way.
What is the scope of the issue? There are half a billion professed charismatics on the planet. He pointed out that we feel great freedom to confront Mormons and Mormonism, though there are merely 14 million of them. Yet we hesitate to address 500 million charismatics.
He turned to Leviticus 10 to explain the name of the conference and the heart behind it, showing true and false worship from Leviticus 9 and 10. The highest duty and greatest privilege of humanity is to worship God and this is always the Christian’s priority. The most serious activity anyone will ever do is worship. When professed Christians come together and say it is for the purpose of worshipping God, they have pronounced upon themselves a seriousness and urgency. Sadly, too much worship today has become trivialized.
The sons of Aaron had been given special privilege and were in line for the high priesthood. They seemed so godly and so secure, and yet God consumed them because they offered strange fire, worshipping in a way he did not sanction. What may have seemed like a minor matter was actually a serious and significant sin. This shows that the most serious crimes against God occur in corrupt worship.
The charismatic movement continually dishonors God in its false forms of worship. It dishonors the Father and Son, but most specifically, the Holy Spirit. Many things are attributed to the Holy Spirit that actually dishonor him. In many places in the charismatic movement they are attributing to the Holy Spirit works that have actually been generated by Satan. Again and again MacArthur stressed the great danger for those who worship God flippantly. It is a tragic and agonizing irony that those who claim to be most devoted to the Holy Spirit are following patterns that blaspheme his name.
He paused to state that he is not discrediting everyone in the movement. He knows there are charismatics who desire to worship God in a true way. Yet the movement itself has brought nothing that enriches true worship. It has made no contribution to biblical clarity, biblical interpretation or sound doctrine. The church had all of these things long before the charismatic movement happened. A Christian today can go back and read the apostles, the Reformers and the Puritans and find richness, understanding and clarity; the charismatics have not added anything but chaos, confusion, misrepresentation and misunderstanding. People have been saved in charismatic churches, but nothing coming from that movement has been the reason they were saved. Nothing within the movement has strengthened the gospel or preserved truth and sound doctrine. It has only produced distortion, confusion and error.
(Note: I am adding a clarifying note (3:57 PM EST). I do not take MacArthur to mean “nothing good has ever come out of the charismatic movement” but “nothing good has come out of the charismatic movement that is attributable to charismatic theology.”)
Yet, though he is grateful for those who do know the truth and are faithful to it, the vast majority are in the dark. His fear is that across the world vast numbers of people in the movement are lost, chasing carnal desires and false gospels. This movement’s appearance of success does not come from its connection to the kingdom of light but the kingdom of darkness.
And despite this, Evangelicalism has thrown open its arms and welcomed this Trojan Horse, allowing an idol in the city of God. This idol has fast taken over.
MacArthur then contrasted Reformed theology with the charismatic movement and said that Reformed theology is not a haven for false teachers. It is not where false teachers reside or where greedy deceivers and liars end up. You won’t go to an association of Reformed churches and find false miracles, visions, prophecies, anointings and other supposed miraculous manifestations of the Spirit. Once experience, emotion and intuition become the definition of what is true, all hell breaks loose.
Tracking closely with John Owen, MacArthur showed what Scripture says about the work of the Holy Spirit and compared that to some of the more bizarre manifestations of the Holy Spirit in the charismatic movement.
He went to Hebrews 10 and the warning there about trampling the Son of God. Over the past couple of decades there have been organizations dedicated to defending the gospel of Jesus Christ. We have also defended the Father against the attacks of Open Theism. But this passage also promises punishment for those who insult the Spirit of grace. We know there will be a hotter hell for those who spurn Christ, but the same punishment is there for those who insult the Holy Spirit. This means we should be terrified to insult the Holy Spirit and vigorous in defending him.
In what seemed to be a brief aside, he called for the restoration of the true worship of the Holy Spirit in the church and said that it is zeal for God’s honor that consumes him here. As he sees and hears this false worship, he feels God’s own pain and wonders why the church won’t rise up to defend the Holy Spirit as it has done with the Father and the Son.
MacArthur concluded by saying we can see in Christ a picture of the perfect work of the Holy Spirit, for the Spirit has committed to do in us what he did in Christ. The Spirit was the constant companion of Jesus; Jesus was conceived by the Holy Spirit, matured by the Spirit, anointed by the Spirit at his baptism, sustained by the Spirit in his temptation, empowered by the Spirit for ministry, filled with the Spirit so he walked in perfect obedience while displaying the Spirit’s fruit, perfected by obedience wrought in the Spirit’s power, raised by the power of the Spirit, and even in his post-resurrection ministry was in the power of the Spirit. The Spirit is to us as he was to Christ. If you want to know how he works in us, look at Jesus. Ultimately, the work of the Holy Spirit is to take corrupted image bearers and to restore in them the likeness of Jesus Christ.
He ended with this challenge: “I will start believing that the truth prevails in the charismatic movement when I see the leaders looking more like Jesus Christ and I see that they really are partakers of the divine nature.”
Next up is Joni Eareckson-Tada with a testimony and, following her, R.C. Sproul.
Please note that in these notes I am giving my understanding and my recollection of what MacArthur said. You will gain a far more accurate picture by watching the video yourself.
I am going to leave the comments open here. Please behave. As always, I will turn them off at or near the 100th comment.

Worshipping at the Creation Museum [challies.com - Informing the Reforming] 2013-10-16 08:23
I never met my father’s father. He died several years before I was born and I knew him only as the mysterious “grandpa,” a tall and powerful figure in black and white photos and old newspaper clippings. But my mother’s father I knew well. He was “Bapa” to his grandchildren, a name bestowed upon him by my brother who, with his privileged position as the first grandchild, had his infantile attempts to say “grandpa” turned into a proper name.
Bapa died many years ago, and one of my final and fondest memories comes from when he lived with my family for a time. My grandmother had died and Bapa was descending into Alzheimer’s, but though his memory was fast fading, he would still engage in conversation and would sometimes take an interest in me. One evening I mentioned my interest in computers and his response made me smile then and now. He said, “Computers are amazing these years. They can add….and subtract…and…” And he could go no further. That was all he had.
He was amazed by computers and knew they had stupendous capabilities, but he had no real knowledge of what those capabilities were. He was familiar with only their most basic functions and knew there had to be much more beyond that.
For some reason I thought of that little episode last week when I was at Creation Museum. Of all the exhibits I saw there, the one I may have enjoyed most was the planetarium. The planetarium is a state-of-the-art theater that allows you to recline and gaze up into “space.” The presentation there is meant to display just some of the beauty and vastness of space and in it all to display the obvious hand of a designer who means to make a statement about himself (and, by comparison, to make a statement about us as well).
Not surprisingly, as I gazed at the stars and planets and tried to wrap my brain around some of the trillions of light years of distance between myself and those vast constellations, I found Psalm 8 running like a track somewhere in the back of my brain. “When I look at your heavens, the work of your fingers, the moon and the stars, which you have set in place, what is man that you are mindful of him, and the son of man that you care for him?”
Thousands of years ago, David gazed up into space with the naked eye and saw the moon and perhaps a few thousand stars. He saw just the smallest part of what God had created in space, an amount almost too small to measure. For all we know, he may have thought this was the complete sum of what God had hung in the sky above, and he marveled at it. His wonder led him straight to worship as his heart rejoiced in the God who could create all of this.
A few days ago I looked at stars billions of lifetimes away and stars so immense they make this entire earth look like a grain of sand by comparison, and I wondered what David would say if he could see what we see and if he could know what we know. What would he say if he learned there are 300 billion stars just in our little galaxy and that there may be another 150 billion galaxies beyond our own? The numbers become too big to calculate and stretch our minds far beyond what we can comprehend or even imagine. The immense power and authority of the Being who created this defies all description and all comparison. And to think that he allowed himself to be clothed in flesh, that he was born onto this little speck in space, that he considered us significant, that he even gave up his life to save us. We can ponder that for eternity and never reach the end of it.
Bapa saw only the most basic functions of a computer and marveled. David saw only the most immediate stars and worshipped. It does us good to gaze into space, whether with the naked eye or with the eye of our latest and greatest technologies. And we will find that the words of the psalms ring true all the more. “The heavens declare the glory of God, and the sky above proclaims his handiwork” (Psalm 19:1).

A La Carte (10/16) [challies.com - Informing the Reforming] 2013-10-16 06:00
Help for Women Under Stress - This book by Randy and Nanci Alcorn is free on Kindle, today only. “Randy and Nanci offer you both the hope and the help you are looking for. They not only help you understand what stress is and how it operates, but give plenty of useful tips and strategies for bringing peace to the chaos of your daily life.” Also, I know it’s an odd title but the book is better than the title: When Will My Life Not Suck? by Ramon Presson is $2.51.
I Hate Porn - There are a lot of people who hate porn, but who still look at it every day. I’m hoping they read this and learn to really hate it.
Vulnerable to Attack - Chuck Lawless: “Based on years of my studying spiritual warfare, here are eight ways I’ve seen leaders allow themselves to be vulnerable to the enemy’s arrows…” Speaking of pastors, here are 10 productivity experiments pastors may like to try.
Killing Jesus - Stand to Reason reviews Bill O’Reilly’s Killing Jesus.
The Teeth of Our Exertions - Here’s some investigation into a great Spurgeon quote: “If sinners be damned, at least let them leap to Hell over our dead bodies. And if they perish, let them perish with our arms wrapped about their knees, imploring them to stay. If Hell must be filled, let it be filled in the teeth of our exertions, and let not one go unwarned and unprayed for.”
Regular Bedtime Helps Behavior - “Overall, children who went to bed at a consistent time had fewer behavior problems than those whose bedtimes varied.” The funny thing is, I think it’s largely true for adults as well.
When we reach the outer limit of what Scripture says, it is time to stop arguing and start worshipping. —J.I. Packer

Clear Winter Nights [challies.com - Informing the Reforming] 2013-10-15 08:09
I do not read a lot of fiction. It’s not that I have anything against a good novel, but more that there is just so much I want to know and so many facts I want to learn, that time dedicated to story feels like it is taking me away from a more urgent pursuit. Or, to hear my wife tell it, I’m just a big snob. Regardless, all the experts say I need to read in a well-rounded way, so I do make way for at least the occasional novel.
Speaking broadly, I see two different kinds of Christian novel. The first begins when an author has a great idea for a story, and, in a desire to make it “Christian,” adds Christian elements to it. In this way the story is primary and doctrine is secondary. The other kind of Christian novel begins when the author has doctrine he wants to teach, and he creates a story as a means of conveying it. Here the doctrine is primary and the story is secondary. In the hands of an especially skilled author, Marilynne Robinson for example, a story can do both of these with excellence.
Trevin Wax has published several books in the past and has just made his first foray into fiction with Clear Winter Nights, a novel that falls into the second category: doctrine taught through narrative. The back cover says this:
What happens when a young Christian dealing with disillusionment and doubt spends a weekend with an elderly, retired pastor? They talk. And no subject is off limits. Clear Winter Nights is a stirring story about faith, forgiveness, and the distinctiveness of Christianity. Through a powerful narrative and engaging dialogue, Trevin Wax shows the relevance of unchanging truth in an ever-changing world.
This is the story of Chris Walker, a young man entering into a dark night of the soul where he finds himself questioning the Christian faith he had once so joyfully professed. As he descends into doubt, he grows hard and skeptical and wanders from all he once held dear. Then he and his grandfather Gil are thrust together for a couple of days and he finds someone who will patiently listen and lovingly provide good answers. Chris’ questions are the questions so many people are asking today, and Gil’s responses are wise, winsome and biblical.
Interestingly, the publisher classifies Clear Winter Nights as Christian living rather than fiction, which shows that this is essentially theology in story, doctrine wrapped in narrative. It succeeds well on both accounts.
Wax is a good thinker and in writing this novel aptly plays both parts—the skeptic and the man of confident faith. He is able to take his readers into sound Christian doctrine but without depending upon answers that are just too neat and too easy. Readers will encounter the basics of Christian worldview and apologetics while learning how to defend Christianity against some contemporary charges. They will also come to understand some of the most important implications of Jesus’ death and resurrection and learn what it means to rely on Jesus through all of life’s peaks and valleys. Wax accomplishes this without being heavy-handed and without ever abandoning his story in order to hammer home a pet doctrine.
Wax is also a skilled storyteller. While he writes compelling narrative, but I found him at his best when creating an atmosphere. He has a knack for simile and comparisons and other elements of writing that work together to create intriguing settings. He made me care about the characters, their stories, their beliefs, and their development. Though this is hardly a tale of intrigue or heart-pounding suspense, it contains a story compelling enough that it easily carries through 160 pages. I cared about the characters enough to be just a little bit sad to have to leave them when the story drew to a close.
Theology in story is a genre that comes and goes in Christian writing and one that, in the past, has been used for good and for ill. I am grateful to see Wax both attempting it and succeeding well at it. Clear Winter Nights is a book, a story, that will encourage the Christian and provide answers to the skeptic. I highly recommend it.

A La Carte (10/15) [challies.com - Informing the Reforming] 2013-10-15 06:30
Here are some new Kindle deals that may interest you: Putting Amazing Back Into Grace by Michael Horton ($3.99); The Justification of God by John Piper ($3.99); Liberating Black Theology by Anthony Bradley ($2.99); Keep Your Head Up edited by Anthony Bradley ($2.99); Glory Road edited by Anthony Carter ($2.99); The Faithful Preacher by Thabiti Anyabwile ($2.99); The Baker (Non-)Illustrated Bible Commentary ($1.99).
God is Faithful - It is good to be reminded again and again that God is faithful. Because otherwise we somehow begin to doubt it.
Punishment and Discipline - “God does not punish His children—He disciplines them. There is an enormous difference between punishment and discipline…”
Removing My Children from the Internet - A father explains why he chose to remove all traces of his children from the Internet.
Surreal Landscapes - There are some amazing and bizarre landscapes in this gallery.
My 9-Year Old Boss - “I got hired at a Bangladesh sweatshop. Meet my 9-year-old boss. Meem, 9, works 12-hour shifts at a factory in Dhaka, Bangladesh. She dreams of becoming a sewing operator, buying more hair clips and helping her family.”
The Ultimate Secret - Here is the ultimate secret (which, of course, is really no secret at all) to effective communication: Hard work.
I was but a pen in God’s hands, and what praise is due to a pen? —Richard Baxter

It's Not Just a Guy Thing [challies.com - Informing the Reforming] 2013-10-14 07:55
Every guy has received a warning about “the second glance.” Here’s how it works: When you see an attractive woman, you are morally responsible for the second glance, not the first. Because you cannot help seeing what is there in front of you, the second glance is the one where you will display sin or virtue. It is here that you make the moral choice—the choice to lust or the choice to direct your eyes and your thoughts to something that honors God.
I have never been completely comfortable with the second glance logic. More on that in a moment, but first we need to see that this is not only a guy thing.
Women can have the same issue or one that is very closely related. For some women the issue is identical—looking with lust. For others it may be something else, such as alighting your eyes on someone who doesn’t fit in and then allowing yourself condescending thoughts about her. It may be thinking unkind thoughts about the immodest woman or the too-modest woman or the woman whose children are dressed so perfectly or so imperfectly. Whether you are a man or woman, you will be tempted at times to allow your eyes to direct you to people who will then take your thoughts in unholy directions. It is a universal problem.
Back to the question: Is it only the second glance that counts? Yes and no.
We have no ability to avoid seeing so much of what we see in the world. At some point we will all see something that feeds one of our great temptations. We will see someone who is dressed wildly inappropriately or someone who is shamelessly intending to draw attention to themselves. And then it is, indeed, the second glance that counts. Will we look again? Will we let our eyes linger? Will we look with lust or condescension or judgment? Will we allow this to be an opportunity to gossip? Or will we immediately and sinlessly direct our attention elsewhere?
But here is where it gets a little more complicated. I think we all know by experience that there is more than one way our eyes can take in the world around us. We can walk into church with our hearts content and enjoying the good things God has given us. We can walk into the mall with our minds submitted to God and eager to please him. And in those times we undoubtedly struggle a lot less with second glances.
But we can also walk into church discontent and grumbling about all we haven’t received. We can stroll through the mall with minds that have not been taken captive by better things. When this is the case, we tend to scan our environment looking for opportunities to lust, to judge, to linger. Our hearts are now in such a posture that we are eager to look at things that are forbidden and we should not be at all surprised when we find them and dwell on them. Just like David on the rooftop, looking for and finding adultery.
The second glance logic assumes that we are going through life in a morally-neutral or morally-upright posture so that there is really no connection between the first glance and the second. The reality is, though, that the second glance has often already been decided long before the first.

A La Carte (10/14) [challies.com - Informing the Reforming] 2013-10-14 06:18
Strange Fire - It has just been announced that since the Strange Fire conference sold out so quickly, it will be live-streamed to anyone who wants to watch it. It runs from this Wednesday to Friday. Would you be interested in having me live-blog it and making this a place we can discuss it? I find myself torn.
Breaking Good - R.C. Sproul Jr.: “With its final episode airing recently we heard an awful lot lately about Breaking Bad, a program that told the fictional story of a high school teacher and family man who becomes a vicious killer and cooker of meth. Like watching a car wreck we all have a morbid interest, to mix a metaphor, in moral crash landings.”
Reformation Art - Reformation Art is having a sale for the next few days; you can get 50% off anything in their catalog.
Imperfect Pastors - I quite enjoyed this article at True Woman on the bravery of imperfect pastors (which, as it happens, was penned by my mother).
Friend of Sinners, No Friend of Sin - “People reviled Jesus. They called him a glutton and a drunkard, a friend of tax collectors and sinners. Have you ever been called names like this? Have I? Do we fear contamination from the world more than we have confidence in Christ’s power to cleanse?”
John Shelby Spong - Here’s a sad article on Spong and his lack of regrets as he looks back on life. “ ‘The older I get the more deeply I believe but the fewer beliefs I have,’” he said, citing an adage once relayed to him by an older bishop. ‘And I think that’s probably where I am. I have a sort of mystical awareness (of God) that’s indescribable, but I can’t avoid it. When I’m asked to define God I’m almost wordless.’”
Microsoft Mission Impossible - I still enjoy reading articles from the world of technology and this one does a good job of describing the challenge to the next CEO of Microsoft.
Intro to Drums - This is a fun and fascinating little video about drums. Yes, drums.
Keep praying, but be thankful that God’s answers are wiser than your prayers! —William Culbertson

The Philanthropists: John D. Rockefeller [challies.com - Informing the Reforming] 2013-10-13 06:30
John Davison Rockefeller was a Christian, an industrialist and a great philanthropist who founded, among other institutions, the University of Chicago and The Rockefeller Institute for Medical Research (now known as Rockefeller University) in New York City.
Born in 1839 to William and Eliza Rockefeller, John was the second of their six children and their oldest son. In contrast to his father, who was known as an unproductive schemer, John gained a great reputation for being an honest, generous Christian.
From his work in the oil industry to his interest in education and science, Rockefeller had a significant, lasting impact on others. In fact, through the trusts and foundations he established, Rockefeller shaped modern philanthropy. Thus, he was the first American worth more than a billion dollars and, when accounting for inflation, many regard him as the richest person who has ever lived.
Though his father was known as a dishonest peddler, he did teach Rockefeller how to earn and keep money. However, it was his mother who taught him to make God central in his life, to pursue integrity, and to give to others. Thus, he once said, “From the beginning, I was trained to work, to save, and to give.”
In 1864, Rockefeller married Laura Spelman and together they had five children. Laura was also a strong Christian and they always made a high priority of worshipping as a family at their local church. His wife was also a wise counselor to Rockefeller. He once commented, “Her judgment was always better than mine. Without her keen advice, I would be a poor man.”
Rockefeller was a conservative Baptist who believed that a strong relationship with God was necessary for honest, meaningful, lasting work. For that reason he always sought to partner with other Christians who shared such views.
As was noted earlier, Rockefeller believed in the importance of generosity. In fact, he tithed ten percent of his first paycheck to his local church and maintained that discipline throughout his life. With his high value on education, Rockefeller helped fund colleges like Spelman College, which was named after his in-laws.
Rockefeller also supported missionary work. In 1905, he gave a grant to the American Baptist Missionaries to help establish Central Philippine University, the first Baptist University in Asia. He also supported Ivy League schools such as Yale, Harvard, and Brown. In 1913, he created the Rockefeller Foundation which gave to public health, medical training, and the arts. This foundation endowed Johns Hopkins and it built the Peking Union Medical College in China.
But he did not merely fund causes. He also invented certain ways of giving that are still used today. For example, he created the conditional grant, which promoted both recurring donations and the opportunity for donors to oversee the mission and value of the organizational recipients.
Rockefeller died of arteriosclerosis in 1937, two months before his 98th birthday. All throughout his life, he knew (in his own words), “God gave me money” and so he embraced John Wesley’s saying, “Gain all you can, save all you can, and give all you can.” Or, as Ephesians 4:28 says, “Let the thief no longer steal, but rather let him labor, doing honest work with his own hands, so that he may have something to share with anyone in need.” Thus, instead of scheming in business as his father had done, Rockefeller worked hard so that he could earn money so that he could in turn give to others.

Weekend A La Carte (10/12) [challies.com - Informing the Reforming] 2013-10-12 06:46
Appreciating Our Differences - There is value in reading this article about appreciating differences in marriage; there may be a bit more value in actually filling out the assessment and talking it over.
A Slow Death to the Soul - I think the best part of Mez McConnell’s article on mercy ministry comes near the end when he says, “It is staggering but true that many churches just haven’t thought about what they are going to do with somebody who comes to faith through their mercy ministry. What is the discipleship strategy? Who will care for them? Who will hold them accountable? How will we move them forward in their walk with Jesus? How will we prepare them for whatever ‘work of service’ God has called them to?”
Gladwell’s Return to Faith - Here’s a short and interesting interview with bestselling author Malcolm Gladwell who describes a return to faith. (He grew up in a Mennonite home not far from where I live.)
Real Men Journal, Too - Writing for CBMW, Ivan Mesa offers seven reasons men should consider keeping a journal.
Harvest 2013 - It’s Thanksgiving weekend up here in Canada, so this harvest photo gallery seems appropriate.
Judgement, Salvation & the Living and the Dead - You may enjoy this sermon jam featuring Dick Lucas.
The Grace of Repenting to Your Kids - Do not be afraid of repenting to and in front of your children; they will respect you more, not less.
God’s heart, not mine, is the measure of his giving; not my capacity to receive, but his capacity to give. —C.H. Spurgeon

Free Stuff Fridays [challies.com - Informing the Reforming] 2013-10-11 10:58
This week’s Free Stuff Fridays is sponsored by Christian Statements, sister company to Missional Wear. They are offering 5 prizes, each of which is a $50 gift certificate to their store.
Christian Statements offers removable decorative wall vinyl with a focus on scripture verses, inspirational phrases and quotes from your favorite theologians. They are ideal for making a statement in your home, church or place of business. You can see their best sellers right here. If you guys aren’t as interested in decor, you may still want to win it for your wife or pastor/ministry leader!
Finally, to celebrate October being Pastor Appreciation month, you can use the coupon code PASTOR to receive 15% off any order this month.

Giveaway Rules: You may enter one time. As soon as the winners have been chosen, all names and addresses will be immediately and permanently erased. Winners will be notified by email. The giveaway closes Saturday at noon.
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The Nature of Christian Zeal [Gairney Bridge] 2013-10-30 07:53
“Truly I say to you, among those born of women there has not arisen anyone greater than John the Baptist! Yet the one who is least in the kingdom of heaven is greater than he. From the days of John the Baptist until now the kingdom of heaven suffers violence, and violent men take it by force.” ~ Matthew 11:11-12
[Christopher] Love described sacred zeal as a violence “opposed to lukewarmness in religion, to that coldness and frozenness that is in the hearts of men under the preaching of the Word.” When asked about the nature of this holy violence, Love said it is “a full and vehement vent of a man’s desires, affections, and endeavors after Jesus Christ in the gospel, so that no difficulties or discouragements whatsoever shall take him off from his pursuit after Christ in the way of His ordinances.” … [B]y nature Christian zeal is the grace that invigorates and inflames all our affections toward a sole purpose and, more specifically, a holy purpose. It is the gracious propensity give to the soul by the Spirit of God that incites and inclines all our affections toward God and His kingdom. It is the divine grace that enables the once barren affections to bring forth the fruits of righteousness in every area of life. Without it, we would make our way to the kingdom at a snail’s pace. Rather than taking heaven by a storm of holy violence, we carry on as if someone will run the race for us; rather than burning with a desire for Christ that refuses to be denied or put off, our affections are as good as asleep, flowing thick and heavy within and hardly moved by anything without. May God rid us of lukewarmness and grant us this fiery zeal.
Living Zealously, pp. 9-10
An Appeal for Zeal [Gairney Bridge] 2013-10-29 08:43
I began reading Living Zealously this morning (I saw a copy on the book table at the Reformation Worship conference, and decided to order a copy last week), and immediately came across this stirring quote in the first chapter:
Who among us cannot see the difference between the early church and our churches, between the apostles and ourselves, between the Reformers and Puritans of several hundred years ago and those of us who claim to be their heirs today? A fire burned in their heart, but are we aflame at all? They seemed driven by a holy passion and resolve, but little seems to motivate us. They were at war with their sin and pursued holiness as if empowered by heavenly strength, but we are too much at peace with our sins and content to do little more than the minimum that God requires of us. Why is there such a difference between them and us?
The difference, according to the authors (Joel Beeke and James La Belle), is Christian zeal. Zeal, says the Puritan John Reynolds, is “an earnest desire of a concern for all things pertaining to the glory of God and Kingdom of the Lord Jesus among men.” The authors of the book lament a lack of zeal among modern Christians:
We lack zeal for God’s honor and glory (1 Cor. 10:31), zeal for God’s house and God’s Word (Pss. 69:9; 119:139), zeal for the advance of Christ’s kingdom (1 Cor. 9:19-23), zeal for repentance and good works (Rev. 3:19; Titus 2:14), zeal to “cut off’ the offending hand and “pluck out” the offending eye (Mark 9:43-48), zeal for that “holiness, without which no man shall see the Lord” (Heb. 12:14), zeal that overcomes all obstacles and perseveres to the end (1 Cor. 9;24-27).
Of course, not all Christians are lacking in this kind of zeal. How about yourself? Do you have a zeal for God’s worship and God’s word? Are you too comfortable in your own sins and too comfortable with the “minimum” that God requires? Perhaps such a work, which examines the sermons and writings of seven Puritans on the subject of zeal, might be a good read for you. Lord willing, I will begin posting some relevant quotations in the coming weeks. May God rekindle our hearts with a holy passion and zeal for His glory!
Mercy Found in Christ Alone [Gairney Bridge] 2013-10-27 17:59
The Gospel, is as much to say, in our tongue, as Good Tidings: like as every one of these sentences be—
Christ is the Saviour of the world.
Christ is our Saviour.
Christ died for us.
Christ died for our sins.
Christ offered himself for us.
Christ bear our sins upon his back.
Christ bought us with his blood.
Christ wash us with his blood.
Christ came in the world to save sinners.
Christ came in the world to take away our sins.
Christ was the price that was given for us and for our sins.
Christ was made debtor for our sins.
Christ hath paid our debt, for he died for us.
Christ hath made satisfaction for us and for our sin.
Christ is our righteousness.
Christ is our wisdom.
Christ is our sanctification.
Christ is our redemption.
Christ is our satisfaction.
Christ is our goodness.
Christ hath pacified the Father of Heaven.
Christ is ours, and all his.
Christ hath delivered us from the law, from the devil, and hell.
The Father of Heaven hath forgiven us for Christ’s sake.Or any such other, as declare unto us the mercies of God.
Patrick Hamilton, from Patrick’s Places
Pitfalls of Preaching [Gairney Bridge] 2013-10-23 09:27
At the recent Reformation Worship Conference, Carl Trueman delivered a presentation entitled “Tips and Pitfalls of Preaching.” Let’s just say it was a lot more about pitfalls than tips! Here are 9 pitfalls he presented, along with some comments he gave:
A Biblical Theology of Worship [Gairney Bridge] 2013-10-22 19:59
This past week I had the privilege of attending the Reformation Worship Conference in Powder Springs, Georgia, held at Midway Presbyterian Church (you can read Carl Trueman’s musings on the conference here).
I found T. David Gordon’s presentation on “A Biblical Theology of Worship” to be especially good (there were two parts to session, but I was only attended the first). In it, he presented a very simple but profound examination of what worship is, according to Scripture. Below are some of my notes taken from the presentation.
Gordon also presented (in a handout) 5 triads that looked at the three different “movements” in human history and how they are re-enacted in Christian worship. These movements can be thought of in different ways (Thomas Boston thought of the movement in four ways!), but most Christians are probably most familiar with the triad of Creation, Fall, and Redemption. In creation, Adam and Eve enjoyed God’s presence; when they sinned, they were banished from that presence; in Christ, we are able to meet with God again. Gordon writes:
To re-enter God’s presence through the mediation of Christ and the power of His Spirit in our weekly assemblies is thus an anticipation of our eschatological return to God’s presence, when: ‘the dwelling place of God is with man. He will dwell with them, and they will be his people, and God himself will be with them as their God’ (Rev. 21:3).
Gordon stated that he hopes to write a book on this topic at some point. If he does, I will look forward to reading it.
A Serpent’s Fruit [Gairney Bridge] 2013-10-22 08:48
Today’s “Making God’s Word Plain Daily Devotional“:
Evidence of False Teachers
“Beware of false prophets which come to you in sheep’s clothing, but inwardly they are ravening wolves. Ye shall know them by their fruits. Do men gather grapes of thorns or figs of thistles?” (Matthew 7:15,16)
When Christ described the working of the false teachers, He was not talking about their characters so much as about the results of their teaching and their policies. Suppose we were to attempt to judge the Lord according to those standards that are accepted in many quarters today. We can well imagine a scene in a Palestinian home during the time Christ was here on earth. Someone would say, “Well this Jesus cannot be a good man, for what He is saying does not make for peace. Now the Pharisees are men of peace. They are such good men. I have seen them praying in the market place. They give tithes of all they possess. They want harmony. But this Jesus is a controversialist. He uses such terrible language. He calls these men ‘generations of vipers,’ and ‘hypocrites.’ Surely we must see that by their fruits ye shall know them, and we shall have to choose the Pharisees.”
And in the days of St. Paul–how easy it would have been to make a case against him! He even called men by name in accusing them of false doctrine. Euodia and Syntyche were to stop fighting (Philippians 4:2). Hymenaeus and Alexander were to be delivered unto Satan that they might learn not to blaspheme (1 Timothy 1:20). Hymenaeus and Philetus were teaching that the resurrection was past (2 Timothy 2:17). Demas had forsaken him (2 Timothy 4:10) and Alexander the coppersmith had done him much evil (2 Timothy 4:14). John wrote that Diotrephes loved the preeminence (3 John. 9).
What false doctrine it would be to interpret “By their fruits ye shall know them ” in such a way as to condemn these Spirit–filled men! Were Paul and John not to be held in honor because they had thus acted? What folly it would be to say that the Pharisees, soft–voiced and smiling, were to be revered above the Christ Who turned over the tables and drove out the money-changers! No! When our Lord said “By their fruits ye shall know them,” He was not talking about the outward appearance of their lives, but about the spiritual fruit that resulted from their work.
Dr. Barnhouse urges that we judge the fruit that the teacher produces not his character alone. A smiling, kindly heretic is no substitute for the firm, persevering servant called and anointed by God.
Certainly this is wise advice, especially the comments at the end which reflect up Donald Grey Barnhouse’s statements. However, something struck me when I considered the part about Jesus’ reference to the Pharisees as a brood/generation of vipers.
The theme of the struggle between the seed of the serpent and the seed of the woman (Genesis 3:15) is woven throughout the Old Testament and even makes its way into the New Testament. Certainly such imagery would not be lost on those who were so familiar with the Hebrew Scriptures, especially the Torah. If Jesus was essentially calling them seeds of snakes, how could they not see the Genesis 3:15 allusion?
The danger of false teaching does not end there. Paul writes these words in 1 Timothy 4:1-3:
Butthe Spirit explicitly says thatin later times some will fall away from the faith, paying attention todeceitful spirits anddoctrines of demons, by means of the hypocrisy of liarsseared in their own conscience as with a branding iron, men whoforbid marriage and advocateabstaining from foods whichGod has created to begratefully shared in by those who believe and know the truth.
These false teachers (who may indeed seem wise in what they advocate), are actually proclaiming doctrines of demons and are therefore instruments of the evil one. This is something to consider in a very sober manner. We must make sure that what we advocate is Christ, and who we listen to proclaims Christ. Otherwise, we run the risk of watering a serpent seed in our hearts — which is far more subtle than we might think.
False Dichotomy [Gairney Bridge] 2013-09-23 10:04
If people have difficulties with God’s judgment here [Genesis 38:6-11], it is, I think, a matter of taste rather than substance. They will likely raise the bogey of the ‘Old Testament god,’ blowing people away for the slightest offense and dropping folks in their tracks for minor slips. But it’s all a smokescreen by folks who don’t read the whole Bible. What do they do with the ‘New Testament god’ who arranges a double funeral because folks fudged about a real estate deal (Acts 5:1-11)? Why did folks at Corinth end up in the ER or the morgue because of a little arrogance at the Lord’s Supper (1 Cor. 11:30)? Why the ‘severity’ of Hebrews 10:26-31 and 12:18-29? ‘Difficulties’ with Old Testament narrative often reveal more about us than about the Old Testament. We tend to get irritated if God doesn’t fit our notions of what he ought to be. We don’t, truth be told, want some God we have to fear. Which is to say, we don’t want the real God.
~ Dale Ralph Davis, The Word Became Fresh, pp. 64-65
I would add to this that such as views as the one Davis is criticizing also overlooks this “OT god” who astonishingly forgives an entire city of nasty folks when His own prophet expected them to be annihilated (see Jonah 3). Jonah himself reluctantly confesses, “I knew that You are a gracious and compassionate God, slow to anger and abundant in lovingkindness, and one who relents concerning calamity” (Jonah 4:2). Indeed, as the prophet proclaims elsewhere (from the most unlikely of places), “Salvation is from the Lord” (John 2:9).
Davis also points to the sobering words of question 84 of the Westminster Question 84: “What doth every sin deserve? Every sin deserveth God’s wrath and curse, both in this life, and that which is to come.” Simply put, we don’t rightly see ourselves as sinners, deserving God’s just punishment against sin, and in our sin, we want to sit in judgment of God. Hence, we play all sorts of games to get around the real need to we have — to repent and turn to the Lord Jesus Christ, the only One who can bring us peace and reconciliation.
The Gracious Mover [Gairney Bridge] 2013-09-04 15:38
From “The Mover,” found in The Valley of Vision:
O Lord, I am astonished at the difference
between my receivings and my deservings,
between the state I am now in and my past
gracelessness,
between the heaven I am bound for and
the hell I merit.
Who made me to differ, but thee?
for I was no more ready to receive Christ
than were others;
I could not have begun to love thee hadst thou not
first loved me,
or been willing unless thou hadst first made me so.
O that such a crown should fit the head of such
a sinner!
such high advancement be for an unfruitful
person!
such joys for so vile a rebel!
BTW, I found this helpful prayer guide for reading (and praying through) The Valley of Vision. May it be useful to others.
The Power of the Good News [Gairney Bridge] 2013-08-15 16:18
Here is my most recent article for the church newsletter at Midlane Park ARP Church:
Just prior to the meeting of General Synod this past June, a pre-Synod conference was held in which attendees were give copies of various books on outreach and evangelism, including one entitled The Secret Thoughts of an Unlikely Convert: An English Professor’s Journey into Christian Faith. What makes this book rather remarkable is that the author, Rosaria Champagne Butterfield, was probably the last person you might think would ever become a Christian. She was an English professor at Syracuse University, and in the book she describes herself as formerly being “a radical lesbian feminist professor.” She was befriended by a Christian couple (in their 70s, no less), who showed her hospitality and spent years sharing the gospel with her. She eventually became a Christian and left her old life behind. Today, she is the wife of a pastor, a home-schooling mom of several children, and a member of an RPCNA church (the Reformed Presbyterian Church of North America – a denomination that is close cousins with the ARP).
Do we think the gospel is for people such as Dr. Butterfield? We often forget the radical nature of conversion. It is not simply a matter of learning new facts about Jesus. Doctrine is certainly important, for the gospel is a message of “good news” (that is what “gospel” means), and we must know who Jesus is and what He has done for the salvation of sinners. But salvation involves faith and repentance. We must place our trust in Jesus alone – only He can save us. And when we come to Christ, this results in is a turning away from the old patterns of sin – a breaking with past sins and old ways of living as we embrace the mercies found only in Jesus Christ. As the Apostle Paul tells us, the gospel is “the power of God unto salvation” (Romans 1:16). It is nothing less than God’s sovereign power that saves us.
This is why we want the gospel to be an emphasis here at Midlane Park. The preaching of the gospel is central to our worship on the Lord’s Day. But the gospel must also be central to our lives outside the church. Currently, we are offering a Sunday School class to refocus our attention on the importance of the gospel and evangelism. We hope to provide other ways to encourage evangelism this fall, including a season of prayer for evangelism, and instruction on how to share the gospel with others. If the gospel truly is good news, then we want to make sure that others hear of this good news.
If we ever forget how powerful the gospel really is, we should remember the story of Rosaria Butterfield. Or, we can remember the story of the Apostle Paul. In 1 Timothy 1, he reminds us that he was formerly a violent persecutor of the church. In other words, he was we might today call a religious terrorist. He persecuted Christians and thought he was actually serving God! But Paul points us to the best news of all: “It is a trustworthy statement, deserving full acceptance, that Christ Jesus came into the world to save sinners, among whom I am foremost” (1 Timothy 1:15). Surely, if this powerful good news was for the “chief of sinners,” it is good news for us as well.
Recommended:
A book: The Secret Thoughts of an Unlikely Convert
A podcast: Unsensational Supernatural Unlikely Conversion
A sermon: Will Your Gospel Transform a Terrorist?
Called As Saints [Gairney Bridge] 2013-07-13 21:30
We sometimes speak of an individual man or woman as ‘a saint’ or refer to ‘St. Peter,’ ‘St. Mary,’ or the like. This is not a New Testament usage. The word is never used there of any individual believer. It is always plural when used of believers, and the plural points to believers as a group, a community set apart for God. Again, the term does not convey the idea of outstanding ethical achievement which we usually understand by ‘saintliness.’ While the importance of right living is insisted on and may even be implied with this very term, the main thrust is not there. It is rather in the notion of belonging to God.
~ Leon Morris, The Epistle to the Romans, p. 53
N.B.: A possible exception to this is Philippians 4:21, which reads, “Greet every saint in Christ Jesus.” The word “saint” is singular in the verse, but the use of “every saint” implies that there is a plurality of saints in the church at Philippi.
Compassion is Underrated [Good News for Toronto] 2013-04-19 14:46
In Colossians 3:12, Paul writes, “Put on then, as God’s chosen ones, holy and beloved, compassionate hearts …” As those united to Jesus in his resurrection, we’re to look like him with resurrected compassion. Now, I know it’s good to be compassionate, but how significant is it?
I often hear Christians express noble desires like these: I want a better prayer life; I want to be more disciplined in my devotions; I want to be more bold with the gospel; I want to overcome sexual sin; I want God to help me with my anger; and so on. Indeed, these are holy ambitions.
But I rarely hear Christians say, “I want to become more compassionate.” Why? Is it too embarrassing to say out loud? Or, is it that we don’t value it much? Or think about it much? Now, I know the excellencies of Christ are endless and any attribute is worthy of meditation, but I can’t help but think that compassion may be underrated. So, I want to behold the compassion of Jesus, with the hope that it will help me to become more compassionate like him, and accordingly, more of a blessing to others. Here are three observations:
1) Compassion is Passive
In Matthew 9:36, “When [Jesus] saw the crowds, he had compassion for them, because they were harassed and helpless, like sheep without a shepherd.” Matthew uses this word for compassion (or pity) five times (Mt 9:36; 14:14; 15:32; 18:27; and 20:34). Four of the five occurrences are in the passive voice, and one is in the middle voice. Who cares? Well, what this means is that compassion is something that happens to you. Jesus sees the lost sheep and is affected. He sees the hungry people (whether it’s 4000 or 5ooo) and is moved. He sees blind men calling out for mercy and his heart feels deeply. This is compassion. It’s passive. It happens to you.
Christ’s heart is soft and tender, easily moved and affected by the plight of man. He sees the lost sheep, harassed and helpless. The sheep don’t have a good shepherd, only selfish and self-absorbed shepherds (cf. Mt 23:5-7) who fail to strengthen the weak, heal the sick, bind up the injured and find the lost (cf. Ezek 34:2-6) . Their interaction with the sheep is described as “harassing.” What do we make of the hearts of the scribes and Pharisees? Their hearts are stone cold, hard as a rock. And even so, the sheep are still responsible, accountable to God, but helpless. Yet God himself, in the person of Jesus, comes to seek out the lost sheep (Ezek 34:11). Jesus is moved by their lost-ness. He knows the danger of the impending judgement (Mt 25:31-46).
2) Compassion is Active
Jesus is moved and affected, yes, but he is moved to action. Notice how the compassion of Jesus compels him: “Then he said to his disciples, ‘The harvest is plentiful, but the labourers are few; therefore pray earnestly to the Lord of the harvest to send out labourers into his harvest’” (Mt 9:37-38). And “These twelve Jesus sent out instructing them … ‘Go rather to the lost sheep of the house of Israel’ … saying, ‘The kingdom of heaven is at hand’” (Mt 10:5-7). Jesus’ compassion compels him to mission and mobilization. He has compassion on the hungry and feeds them. He has compassion on the blind men and heals them. He has compassion on the lost and calls his disciples to pray for more workers and to go preach and heal. Jesus’ compassion is active. His compassion compels him to action.
3) How to Cultivate Compassion
Looking at the compassion of Jesus highlights my problem. I lack compassion. I see the tenderness of his heart, but it convicts me of the hardness of my own. Why am I not more easily moved and affected by the plight others? Is their hope for my heart? What can I do?
In the context of Matthew 8-9, we see Jesus’s authority over leprosy, paralysis, sickness, demons, nature, sin, and even death. Matthew wants us to know that Jesus has authority over all things; this includes our hearts! He has the authority to say, “Let it be soft.” So, there’s definitely hope. But let’s think about what we can do. Here are 5 ways to help cultivate compassion for the lost:
1) Look at the compassion of Jesus. This kind of beholding has transformative impact (see 2 Cor 3:18).
2) Consider the value of people. Jesus saw the value of people, created in the image of God, and thus precious and loved by God (Isa 43:11). He wasn’t indifferent to anyone.
3) Move toward people & learn their story. It’s easy to move away from people, especially different and difficult people. But since people are precious, Jesus moves toward them. He entered our world, became a man (Phil 2:5-11) and identified with our sufferings and temptations (Heb 2:10; 4:15). He moves toward people. And he knows our stories. Think of Jesus’ interaction with the women at the well (John 4:1-26). He knew how sad and sinful her life was; yes, he knew her story, the exact number of husbands she’d had. But he moved toward her, not away. He listened to her, spoke with her and helped her. He knew the stories of the crowds of lost sheep. He knew all about the “harassed and helpless.” He often had dinner with sinners and tax collectors – even in their homes! I can certainly envision Jesus asking people about their lives as he showed them their need for redemption. He never just listened; he was also always affected.
Often times, the simple act of moving toward a person helps cultivate compassion. As we ask questions and learn about a person’s life and what’s important to them, we better position ourselves to God’s softening power over our hearts. When it comes to moving toward the lost, we must see their story through the theological lens of needing redemption, and as those who are precious to the Redeemer. This will help us to see people the way Jesus does.
4) Remind yourself that you are nothing. Often times, we think we’re something. This is the sinful pull within all those making spiritual progress (Gal 5:25-6:5). But it’s simply not true. We are not “something.” Yes, we’re precious to God, but our value ought to be attributed to God. And our spiritual progress ought to be credited to the work of the Spirit. Paul writes, “When anyone thinks he is something, when he is nothing, he deceives himself” (Gal 6:3). And what does this have to do with compassion? Well, pride says, “I’m something” yet fails to see (and own up to) personal weakness, sin and failure, consequently finding it hard to sympathize and bear burdens (Gal 6:1-2).
So, let us remind ourselves that we are nothing. It will help prepare our hearts to be more easily affected by others. This mindset will also positions us in the same boat as the lost, seated nice and close, where we can compassionately guide them.
5) Pray to become more compassionate. Jesus said, “Ask, and it will be given to you … If you then, who are evil, know how to give good gifts to your children, how much more will your Father who is in heaven give good things to those who ask him!” (Mt 7:7-11). Is asking for more compassion not a good thing?
As those raised with Christ, may God help us to put on compassionate hearts. Amen.
What does Christianity have to do with my life? [Good News for Toronto] 2013-04-17 14:49
Last year, Power to Change (York) asked me to speak on this question: What does Christianity have to do with my life? I answered in two parts. The first can be found on my blog post entitled What is Christianity?, and what follows is the second part of my answer.
I want to highlight two gripping ways that Jesus Christ profoundly relates to you. I found these themes in the Bible; specifically in the ancient biography on Jesus written by his close friend Matthew.
1) Jesus is your judge.
Though many people don’t like the idea of judgement, Matthew’s biography clearly shows that Jesus talked a lot about judgement. In fact, Jesus taught that one day in the future, he will come and judge everyone who has ever lived.
In Matthew 25:31-46 Jesus paints a picture of what Judgement Day will look like. He is the judge. All peoples will stand before him; and he will separate them, the sheep on his right, and the goats on his left. And their sentence? The sheep (the righteous) will hear: “Come, you who are blessed …” But the goats (the unrighteous) will hear: “Depart from me, you cursed …” Jesus is the judge. He is my judge. He is your judge.
In Matthew 7:24-27 we learn that everyone’s destiny is determined by how they respond to the words of Jesus: destruction for those who don’t trust him, taking him at his word; and life for those who trust him, proving their trust by doing what he says. According to Jesus, your destiny is determined by how you respond to him.
The inevitable event of Jesus’ future judgement ought to inform the way we respond to Jesus now. People save money now in light of future retirement. People pick academic programs now in light of future career plans. Future events have direct ramifications for how we live now. How much more the future judgement!
You may be thinking, “but I’m a good person; I’ll be okay on Judgement Day.” I don’t doubt that you’re a nice person; many Canadians are nice. But when we speak of judgement, the only person’s estimation of your goodness that counts is the Judge’s. So, what does it mean to be good according to Judge Jesus? The Sermon on the Mount (Matthew 5-7) is a great summary of his standards, that is, of true goodness and righteousness. He’s not only concerned with what we do, but what we think, and why we do the things we do; he cares very much about motives. A summary of these righteous standards is found in Matthew 5:48, “You therefore must be perfect, as your heavenly Father is perfect.” Truth is, he knows we’re not. In fact, in the sermon, he calls us “evil” (Mt 7:11). Not the most flattering pre-trial assessment. We need a Saviour. We need forgiveness. This brings me to my second point.
2) Jesus has the authority to forgive your sins.
The name Jesus means God saves. Before his birth, and angel came to Joseph in a dream, saying, “[Mary] will bear a son, and you shall call his name Jesus, for he will save his people from their sins” (Mt 1:21). Judgement is coming. We deserve to be punished for our sins, but Jesus came to save us. Christianity is pretty simple; as John Stott taught, it’s a “rescue religion.”
In Matthew 9:12-13, Jesus says, “Those who are well have no need of a physician, but those who are sick … I came not to call the righteous, but sinners.” Jesus came to save sinners. He came to save people – people who realize they’re not good in God’s eyes and yet call out to him for help. He came to save the spiritually sick from the impending judgement that is coming.
The good news is that Jesus truly does have the authority to forgive all of your sins. Anyone, no matter how corrupt your past (or how depraved your current habits), can find full forgiveness in Jesus. Just as he displays his authority over nature, disease, demons and even death, he shows his authority to forgive sins (Mt 8-9). In Matthew 9:1-8, he heals a man who is paralysed and proclaims his authority to forgive sins. Interestingly, he shows his authority empirically while proclaiming his authority to do what can’t be seen: forgive sin. The question is: do you believe? Do you believe he has the authority to forgive sin? Even your sins?
When Jesus came, through his death, resurrection and ascension, he ushered in the New Covenant. A covenant is when God establishes an arrangement with people whereby he is their God and they are his people. When it comes to the New Covenant, Matthew wants us to know that Jesus gave wine to the disciples, saying “this is my blood of the covenant, which is poured out for many for the forgiveness of sins” (Mt 26:28). God is establishing a relationship with people through Jesus Christ wherein people receive the forgiveness of all of their sins by faith in Jesus. As people forgiven by Jesus, God is their God and they are his people. Again, the way into this covenant is not by works or trying to be a good person. It is by faith in Jesus Christ. This is the only way to receive forgiveness; and therefore peace with God. Everyone from every nation and background is both commanded and invited to come to God through faith in Jesus Christ, the only one who has the authority to forgive sins.
How else will you receive forgiveness?
Though I could go on, those are a couple of pretty significant ways that Jesus Christ relates to you (and me!). You might not feel the relevance; but Jesus certainly sees it. May he help us to both see it and feel it.
What is Christianity? [Good News for Toronto] 2012-10-30 21:47
It was a true joy to speak at a Power to Change event at York University last month. I’m really encouraged to see the way they’re engaging unbelievers with the grace and truth of Christ. I was asked to speak on this question: What does Christianity have to do with my life? Good question. There are hundreds of ways to answer the question, but what follows is the first part of how I responded. I’ll write another post on the second part.
We can capture the essence of what Christianity is from two angles.
1) “Follow Me”
Christianity can be summed up in two words, “Follow me.” These are the words of Jesus and this is the essence of Christianity. Christianity is not primarily a system of beliefs. Though it contains a system of beliefs, it is, in the first place, knowing the person of Jesus Christ and following Him. Jesus’ call to follow him is his call to trust him and prove that trust by doing what he says. Believing that he is who he says he is and that he will do what he says he will do. It is a call to love him, know him and follow him.
2) Christianity is built upon the truthfulness of Scripture, which is the lens through which Christians see the world.
The Bible governs a Christian’s beliefs about origins, meaning, identity morality and destiny. Christianity is founded on the truth of the Bible. The Bible is one story that reveals who God is, but the storyline is composed of four different stages.
1) Creation. God created the world and everything in it. He created it good. He created us good. He made us to live in his presence, under his rule and to enjoy him.
2) Fall. Adam and Eve rejected the rule of God and because God is just they suffered for it. According to God’s just dealing with rebellion, he cast them from his presence. Mankind was damned and doomed. The world was cursed.
3) Redemption. This is God’s a rescue plan; his peace making plan for his enemies. Redemption is what God has done to save rebels from their rebellion and to bring them back to himself to live under his rule, in his family, fully forgiven for the bad they have done. He sent Jesus to save people from the curse and their sins.
4) New Creation. This is the hope of what God has promised. Not only did Jesus come and die and rise and leave. He’s promised to return and judge the world. All rebels who are saved by Jesus will be with him, in his presence, living under his rule, forever. This is the hope of Christianity.
These four stages of the story of the Bible form the lens through which a Christians see the world. But Christianity is primarily about knowing and following the person of Jesus Christ. Jesus not only believed in and confirmed the truthfulness of Scripture, creation, the fall, redemption and new creation, He is the climax and main subject of the entire story. Everything else in the story before him foreshadows him. Christianity is about Jesus.
So now, in light of all of that, so what? What does all of that have to do your life? (Stay tuned for part 2).
“What I’m Offering You Is a Steak!” [Good News for Toronto] 2012-10-24 14:46
The following post was written by my good friend, Alex Philip. Below he gives an encouraging account of a couple of conversations he had this past Friday night.
On Friday, I joined Paul for an evening of evangelism. Paul asked if I would take the lead with another brother from Westminster. I was filled with gladness when this other brother walked into the Tim Hortons: I had taught this young man almost five years ago when he was in grades 10 and 11. Today, he is deliberately seeking to honour Jesus by declaring his gospel to others.
Thanks be to God, we had a fruitful evening with many meaningful and pleasant conversations. There are too many to list in a blog post. Two, though, stick out. The first was with a couple of young men who were clearly heading out for a night of revelry. We approached them with outstretched hands. They refused to reciprocate, insisting instead that we tell them what we were selling…
“We’re not selling anything. We’re giving something away.”
“And what’s that?”
“The opportunity to know that your sins can be forgiven through Jesus Christ.”
The more vocal of the two laughed jeeringly and heartily, saying, “I knew you were pedalling religion.” He continued, “Tell you what, we’re on our way to pick up smokes and then we’re heading back to my apartment where we have a couple of girls…” He proceeded to invite us back to his apartment to join them in their activities.
Realizing that time was short before he and his friend walked away, we declined his invitation saying, “What you’ve offered me is a marshmallow. What I’m offering you is a steak.” He thought the response was funny and retorted, “Okay, you got me. I’ll take what you’re giving away.”
We gave him a gospel tract and before leaving, he finally did shake our hands and give us his name. The last thing I saw was him reading the tract as he headed towards the store to pick up his smokes.
Later on that evening, Kathy and I had a long and meaningful conversation with a young girl who was spending the evening sitting on a bench, listening to her iPod. She was attentive and receptive to the gospel to the point that we were able to present the gospel to her twice. Once from the Bible and the second time from the 2 Ways to Live booklet. At the end of our conversation, we invited her to place her trust in Jesus Christ.
She expressed a sincere concern for the countless number of people who would never hear what she had just heard. Gently, we tried to respond biblically to her concern. We had the opportunity to pray with her on the street corner and she indicated that she would like to come to church. Please pray that she would come and be converted and then enter into fellowship with Christians.
I Asked Him, “What Do You Think of Jesus Christ?” [Good News for Toronto] 2012-10-17 09:55
On Friday night I was at the bus stops at Lawrence and Don Mills with fellow workers (from New City Baptist and Westminster Chapel). Meeting much indifference and hostility made it a sobering night. What follows is a short summary of one of those meetings.
I went up to a big guy who looked around 50. I opened my mouth and said, “Sir, I have a question for you,” and with a gentle smile, “but just to warn you, it’s a serious one.” He looked at me, waiting to hear what it was. “What do you think of Jesus Christ?” With much anger and resistance he said, “Not much.” I quickly responded, “Why?” With more emotion now and added volume, he said, “No more questions! You had one. That’s enough. Thank you.” We stood there silently for a bit. He looked very angry. I said, “Have a good night.” He replied, “You too.” I walked away.
Phil and I reflected on the exchange as we walked. Phil said, “That shouldn’t surprise us; people hate Jesus. The Scriptures are clear about this. Who knows the hypocrisy he’s seen in his life? But, either way, people hate God.” Afterwards, Phil prayed for him.
Let me pray for this man again. Father, please cause that man to reflect on my question, “Why?” Please put that question to his heart: “Why don’t I think much of Jesus?” If it’s because of the sinful conduct of Christians, please help him to see the foolishness of rebelling against Jesus because of what someone else has done. May he see that Jesus is always good. Lord, have mercy on him!
Nick Speaks of Jesus on the Streets: The Story and the Lesson [Good News for Toronto] 2012-10-11 20:14
Nick is the lead worshipper at GFC Don Mills, but more importantly, he deeply loves Jesus and is committed to following him. Below he shares a really cool story about his experience of speaking about Jesus when he came out with me two weeks ago.
Friday night
Paul, Ricardo and I went out on Friday night to speak to people about Jesus. As is Paul’s custom, we walked around different bus stops to speak with anyone who was willing. We had a few good conversations but one that stood out to us was with a young man. Let’s call him Tim though that’s not his real name.
We met a man at the bus stop
Paul and I approached Tim (Ricardo stayed behind to pray) and told him what we were all about. We introduced ourselves by name and informed him that we were members at a church in the area and wanted to speak with people about Jesus. Paul began by asking him if he had any religious background. He told us that his mom was a Christian but he characterized her devotion as “moderate”.
Asking questions
Then Paul asked Tim about himself a little bit. “What are you doing?” Paul said. He responded by telling us that he was on his way home from work. Paul continued, “What kind of work do you do?” “Customer service,” Tim responded. At that point I broke my way into the conversation. “Do you get yelled at a lot?” I asked. “Yeah,” Tim laughed. I pushed the conversation a little further, “In this line of work do you see how messed up people can be?”
So began our conversation.
People are messed up
Tim agreed that some people are indeed messed up; but then he said that some people aren’t all that bad. In fact, he said that some people are good. In some ways Tim was right. People are made in the image of God and they do retain some of the good-ness that God created us with; but I told Tim that it’s interesting that you usually never meet anyone who thinks they are bad themselves. It is usually “other people” who are the problem. He agreed at that point and said, “Yeah, we tend toward an ‘us vs. them’ mentality.” I wanted him to see that “we” are all part of the problem and not just “them,” so I asked him, “Tim if the world was filled with people just like you, do you think that all the world’s problems would disappear?” He said, “Probably not.”
The story of the Bible and its climax
I proceeded to tell Tim the story of the Bible and the climax of that story. It went something like this: Our world has been utterly destroyed by sin and we need a King who can put the world right. God promised that one day he would send his King and this is exactly what we read about in the gospels, Matthew, Mark, Luke and John. Jesus came into the world, in fulfilment of God’s promises, pushing back against sin and all of its effects. That’s why we see him healing diseases, forgiving sins, calming storms, casting out demons and even raising the dead. Jesus eventually died on the cross and rose again so that we ourselves could be forgiven of our sin and included in God’s kingdom. One day Jesus will return and make everything right. The thing about it, though, is if God is going to make the world right and rid the world of sin something has to be done about us. That means either forgiveness or judgement.”
The challenge
Afterwards, Paul challenged Tim. He asked him what he thought about this and if he ever thinks about these things. Tim told us honestly that he has always been indifferent to Jesus. He said that he’s indifferent towards a lot of things.
Our conversation continued for a while (he let three buses go by while we were speaking). He asked some good questions regarding the centrality of God and the importance of the gospel of Luke (We had copies that we were giving out). We let him know that the gospel tells the story of Jesus and is a good place to start but that the entire bible is God’s inspired word. Paul even had the chance to speak with him about Genesis 1-3.
This was an encouraging encounter. Tim was interested, open and honest. I don’t know if I’ll ever see him again but I hope that he bows his knee to King Jesus and is welcomed into the life of the age to come. Pray for him.
Five lessons learned:
1) Talking about Jesus brings us joy: As I was speaking to Tim about Jesus I found that I myself was getting excited about Jesus. I began thinking, “Wow! This really is good news!”
2) Seeing the gospels as the Gospel makes Christianity incredibly relevant to people: There are many stories in the gospels about all sorts of people from all sorts of different walks of life. In Tony’s case he works in customer service. This is a career where one is continually yelled at and made to feel small. There are plenty of stories in the gospels about people who are “made to feel small” and how Jesus meets those people where they are. Ultimately, we see how Jesus came into the world to bring God’s kingdom and solve the systemic problem of sin that causes all the problems we encounter.
3) Just be yourself and talk about Jesus: Evangelistic “schemes” can be incredibly helpful at times (I know I’ve benefited from them at times) but it’s important to just be yourself. We are talking to human beings not robots. Talk to people where they are at and then talk about Jesus. You don’t need a PhD in Missions to do that. Although evangelism can be hard work at times it can also be really fun when we are ourselves.
4) Pray: God is in control and can soften people’s hearts. We need to plead with God that he would do just that and that he would lead us to those whom he wills.
5) Worship: Evangelism is worship. Whether you experience a Tim or a person who wants nothing to do with you God is glorified when Jesus is proclaimed.
What Are You Living For? [Good News for Toronto] 2012-10-10 13:42
One month ago marked our first Friday evening on the streets for the fall. John and Sidney, from Westminster Chapel, joined me for what turned out to be a very encouraging evening. But there was one conversation in particular that I’d like to share with you.
We met two young teenage girls close to the bus stops. It was great to hear Sidney share the gospel with them. The girls listened to her speak of Christ. Sidney told them why Jesus came and about the forgiveness and life that he offers. She did an excellent job sharing the good news about Jesus.
And how did the girls respond? Well, they just looked at us smiling and giggling. They seemed terribly awkward and unsure of how to respond. We thought we’d try to ease the awkwardness by asking questions, hoping that two-way discussion would blossom. Their answers were quick and simple; and they kept giggling. One of the girls knew a little bit about Christianity. She said she’d been to a Christian youth event with her relative. The other girl did not seem to know much about the faith.
I sensed they were just waiting for the conversation to end, but not because they were angry; they just didn’t know why this stuff was important. And it was a Friday night! Why talk to strangers about Jesus? Sounds a little weird. I sensed the need to cut right to the heart. We were real people talking to real people about a real Judge and Saviour! We were talking about stuff that really mattered. So, I cut to the chase and interjected: “What are you living for?”
The one girl looked at me, thought for a moment and said, “High school.” I said, “Why?” She said, “To go to University.” I said, “And I’m assuming in University, you’ll live to do some more schooling; but what for?” She said, “To get a job.” And then I said, “You’ll get a job and then you’ll live to find a husband. And then you’ll live for your kids and your work. Then you live for your kids to go to University. And then you live for your retirement and then you’ll die.” They were silent and listened closely.
I pleaded with them, “Look nothing is more important than Jesus Christ. He said that one day you will stand before him and give an account of your whole life; and all that matters is how you’ve responded to him. That’s pretty serious stuff. It’s not the kind of stuff you should put off. Take your Bible and read the four Gospels that are all about Jesus. Look into these things. Don’t put it off.”
We said our goodbyes, but I couldn’t help feeling utterly refreshed and sobered by getting to heart of things. When we’re out on the streets talking to people, we’re talking to real people about a real living Saviour. Let’s get real and talk, not religion class style, just trying to get the answer right. Let’s talk about what we’re really living for; and why living for the Lord Jesus Christ is all that matters.
Day 21: Let Me Be Clear [Good News for Toronto] 2012-10-04 21:14
Just over a month ago marked the final day of our summer street evangelism. The Lord gave me a wonderful gift: a long conversation with a teenager who had taken a break from his skateboarding. He was relaxing in the shade and seemed open to talking. We talked for a long time about a number of things, but I can still remember how we transitioned to talk about spiritual things. You see, though I’m usually very direct from the outset with what I’d like to speak about (Jesus), this time I wasn’t. I just asked him questions about him; first about skateboarding and then, as the conversation took many twists and turns.
Often times people love talking about themselves, so I just kept asking him questions and got to know him pretty quickly. I learned of his passion and love for art. We talked about art for a while. I couldn’t help but ask him about beauty. How is it that we can appreciate it? Where does it come from? So I asked, “Have you ever thought much about where beauty comes from? I’m a Christian and I really appreciate the beauty in our world. I think it reflects the beautiful mind of the One who created it. What do you think?”
He acknowledged a higher power and embraced some elements of a theistic worldview, but his understanding of Jesus was certainly minimal and incorrect. I shared a bunch of Scripture with him and tried to explain the Gospel to him, but as we conversed, I noticed that I kept having to say, “Oh, well let be clear; I’m not sure if I was clear enough earlier, but …” The main truth that he didn’t seem to get was sin. I had been trying to teach him that we are so sinful that we cannot redeem ourselves; that we’re in need of total repair by someone else; that we’re guilty, evil and without hope in the world and in utter need of a Saviour.
He was a very agreeable person. He seemed to agree with everything I said! It’s tough to share the Gospel with people like this (but may God help us!). When I asked him questions to check his comprehension one thing became increasingly clear, he didn’t really agree with me. Maybe I wasn’t clear. Maybe he was distracted. Maybe he’s not a good thinker. Maybe it’s just the blinding effects of sin. Maybe he’s so steeped in relativism that he can agree with the truth and yet disagree at the same time! I don’t know. But the main thing I can remember from that conversation was having to say, “Let me be clear.”
I was deeply impressed by the difficulty of ministering to the Agreeables. They just agree with you. But do they really? I doubt it.
One of the lessons I learned from that conversation, and that I have seen many times in evangelism, is the need to ask repeat direct questions of the people we’ve preached to. For example, you can talk to a guy about righteousness, sin, guilt, judgement and grace and it may seem that he has an understanding of some of these core truths. You can tell him that salvation by works (or even grace + works) is insufficient (and only further condemns!). And he might agree with you. But then ask him, “So, if you were to die today, do you think God will accept you or reject you?” And what will you hear him say? Sadly, and too often, “I think he will accept me.” And you can follow up, “Why?” Sadly, and too often (at least, among the people I speak too), you’ll hear these words, “We’ll, God knows I trying to be a good person, and he’s forgiving.”
May the Spirit give understanding and conviction! And may He give us endurance to keep preaching! God can save Agreeables! But may He give us wisdom to show them what they really don’t agree with. Direct questions (even if repeated many times) can help.
Day 20: Laughed At [Good News for Toronto] 2012-09-20 15:22
Our second last day of summer evangelism wasn’t without challenges. Ricardo and I sought to speak with a young lady waiting for her bus. I told her who we were, where we were from and that we were out sharing the Gospel. She instantly felt awkward and started laughing. I encouraged her that these are important conversations to have. By God’s grace she was too nice to dismiss us. She engaged in a conversation that ended up recruiting a small measure of mockery (and some more laughing).
What follows is an abbreviated summary of what happened. Y refers to the lady; P refers to me; and M refers to another lady who joined in.
P: What do you think of Jesus?
Y: I don’t know. (And looking at me as though this was a silly question).
P: Well, what do you know of him? Surely you have thoughts of some kind.
Y: Well, I don’t really believe in God.
P: Why do you hold to atheism?
Y: I just grew up with it and I’ve always believed it.
P: Well, in light of the world we observe, do you think it’s a more reasonable position?
Y: I don’t know.
P: Do you believe in the big bang?
Y: Yes.
P: Well, why do you opt for order coming from chaos over the idea that God created everything with order?
Y: Well, I’m not saying it’s impossible. I just don’t believe.
P: Well, have you ever read the Bible?
Y: Not really. But the Bible is not reliable.
P: Why do you think that?
Y: It was written by men.
P: So you’re saying it’s corrupted because it was written by men and men make mistakes?
Y: Yes.
P: But think about this: you are a person. So how do you know you’re not making a mistake in your judgement of the Bible. You said men make mistakes. (She seemed to understand my argument and her body language indicated that she conceded).
P: And surely it wouldn’t be too hard for an all powerful God to keep his Word pure, even through men. (She agreed on this theoretically).
P: The Bible is trustworthy and it clearly teaches that God created the world out of nothing, that he created everything good and that we are the ones who’ve messed up the world. We’ve sinned by disobeying God’s commands. And now we’re not good. And that’s why we feel guilt. You ever feel guilt?
Y: Yes (smiling with the look of, “Why are you talking about this?”).
P: Me too. You know, the reason we feel guilt is because we have it. It’s that simple. It’s real, but that’s the very reason why God sent Jesus into the world. You know about the Cross? How Jesus died on the Cross and rose from the dead? The reason he came to do that was to save us.
Y: Wow. This is intense. (smiling)
M: (Shouting from about 12 feet away, and with all manner of mockery), “What Jesus are you talking about?”
P: (I looked over at her and was silent for a few seconds, and with firmness I said back to her) The Jesus of Scripture that even non-Christians and liberal Bible scholars don’t deny existed.
(Though M was silenced, Y and M were now both laughing with each other at me).
P: (speaking to Y) Look, the reason we’re out talking to people is because these things matter and we care for you. Thank you for taking the time to let us speak to you. Take care.
At that moment Y walked over to M and the two became friends. We could overhear them laughing together and M saying how she needed to “save” Y from me.
By the grace of God, Ricardo and I prayed for them. And by the grace of God we truly considered ourselves blessed. But I really felt opposed spiritually. I felt little and despised by people. I was laughed at. I felt, in the smallest measure, something of what my Saviour felt. And it was good.
Day 19: I Blew It! [Good News for Toronto] 2012-09-19 11:28
Exactly a month ago, I was at a bus stop talking to an elderly man about God. He told me that he’s not really into the Bible but that he’s got his own version of God (and that he’s quite comfortable with that). He was a kind man and quite fond of God’s kindness to him. Let me explain.
God’s kindness
He assured me that he’s on good terms with God. He told me about a couple of serious accidents that he endured and, with God’s help, fully recovered from. He acknowledged that it was God who kept him and helped him recover. As far as he was concerned, he didn’t need God’s mercy; God’s smile was upon him.
Time for me to speak
I listened to him for a while. And after he shared, I sensed the boldness of God to share some words from Jesus. I wanted to share what Jesus said about God being kind to the wicked. Indeed, God’s kindness is not a full proof indication that one is on good terms with God. So I told him, “You know what, everything you’re sharing makes me think of a passage of Scripture I read recently. It’s in Luke’s Gospel. Just one second. Let me find it.” (I pulled out my Bible and starting flipping around in Luke). I stood there searching and searching and searching. I keep telling him, I’m gonna find it; just one minute. This went on and on and on. No joke, I think at least 3 minutes passed! I felt like an idiot. I couldn’t find it! Where is that verse! The bus came. We said our good bye’s and that was it. I didn’t tell him anything!
I blew it!
I had the perfect chance to speak the Word of Christ into his life, but I didn’t have it memorized; and worse, I couldn’t find the address! I failed. This is especially bad because I had been struck by these words of Christ just beforehand and assumed I’d remember them! Bad assumption. So afterwards, I found the passage, and drilled it into my head, saying over and over (aloud), “Luke 6:35, 6:35, 6:35!”
In Luke 6:35, Jesus says of God, “he is kind to the ungrateful and the evil.” Those are some pretty startling words to deal with. You should memorize this reference with me. Countless unbelievers appeal to God’s kindness to them as an indication that they are certainly in his good books. Well, though they may think this way, Jesus certainly doesn’t. God is kind to all. Assurance of being in his good books must be determined another way.
But the lesson to be learned here is this: memorize the reference!
Though I blew it, I know that man’s eternal welfare doesn’t hinge on my performance. May the Lord be kind to him with the loving-kindness of salvation.
We're Moving... [Words of Comfort -- Ray Comfort's Blog] 2012-09-11 14:20
Democrats and God [Words of Comfort -- Ray Comfort's Blog] 2012-09-06 09:20
The Democratic Party’s flip-flop and the resulting anger and confusion about whether or not to put reference to God into their platform, highlighted the great weakness in our political system. The Yes and No vote is reminiscent of another vote that was taken 2,000 years ago, which resulted in the one-man steering committee washing his hands of the whole process.
This is what happens when a nation abandons the Bible as its moral guide. It has no rudder in the stormy sea of human opinion, so it is driven by whatever wind blows the hardest. When the Bible is abandoned, the question is asked, “Is killing a child in the womb morally okay?” The answer comes from those who yell the loudest. The same applies with homosexuality and other moral issues. Abraham Lincoln once said, “Our government rests in public opinion. Whoever can change public opinion, can change the government…” That becomes a weakness and not strength, when the Bible is left out of the equation.
The Scriptures are “a lamp to our feet and a light to out path.” When they are ignored we are left in moral darkness and we will soon forget the truth of the words “God mend thine every flaw, Confirm thy soul in self-control, Thy liberty in law!” Thomas Jefferson said "The greatest danger to American freedom is a government that ignores the Constitution." I propose a small amendment to his words. They should read, "The greatest danger to American freedom is a government that ignores the Scriptures and treats the Constitution as though it was the Word of God."
This fiasco should also remind us of Lincoln’s, “Sir, my concern is not whether God is on our side; my greatest concern is to be on God's side, for God is always right.”
Hospitals Kill Two Million in Ten Years [Words of Comfort -- Ray Comfort's Blog] 2012-09-05 09:30
Get Here, Now! Mr. President [Words of Comfort -- Ray Comfort's Blog] 2012-09-04 09:30
Jeff The Heckler [Words of Comfort -- Ray Comfort's Blog] 2012-09-03 09:30
Yesterday at Huntington Beach:
Darwin's Gift to Humanity [Words of Comfort -- Ray Comfort's Blog] 2012-08-31 09:30
Knuckle-dragging Narrow-minded Creationists (part 2) [Words of Comfort -- Ray Comfort's Blog] 2012-08-30 09:30
(read part 1 here)
But you are not alone if you believe in God. Many of our greatest scientists believed in the existence of a Creator: Galileo, Newton, Nicholas Copernicus, Francis Bacon, Michael Faraday, Louis Pasteur and Kepler, just to name a few. Einstein rightly said, "Science without religion is lame; religion without science is blind." He also said, "In view of such harmony in the cosmos which I, with my limited human mind, am able to recognize, there are yet people who say there is no God. But what really makes me angry is that they quote me for the support of such views."
The incredible harmony in creation proves beyond a doubt to any thinking mind that there is a Creator. Listen to what the Bible says about atheists and the existence of God: “For since the creation of the world His invisible attributes are clearly seen, being understood by the things that are made, even His eternal power and Godhead, so that they are without excuse…” That’s from Romans 1:20.
But God doesn’t want you to just believe in His existence. He wants you to obey Him. When I understood that I had violated God’s Law (the Ten Commandments), and 2,000 years ago Jesus paid my fine in His precious life's blood, I found that upon my repentance and faith in the Savior God would legally commute my death sentence. My case was dismissed through the mercy of the Judge.
On the Day of Judgment the moral Law cannot condemn me, because my crimes against God no longer exist. They have been blotted out by the grace of God. I have everlasting life. My fine was paid; and my case dismissed. Is yours?
Knuckle-dragging Narrow-minded Creationists (part 1) [Words of Comfort -- Ray Comfort's Blog] 2012-08-29 09:30
Homeschoolers Miss Out on Sexual Promiscuity and Much More [Words of Comfort -- Ray Comfort's Blog] 2012-08-28 09:30
Potiphar’s Wife [Words of Comfort -- Ray Comfort's Blog] 2012-08-27 09:30
And it came to pass after these things that his master’s wife cast longing eyes on Joseph, and she said, “Lie with me.” But he refused and said to his master’s wife, “Look, my master does not know what is with me in the house, and he has committed all that he has to my hand. There is no one greater in this house than I, nor has he kept back anything from me but you, because you are his wife. How then can I do this great wickedness, and sin against God?” So it was, as she spoke to Joseph day by day, that he did not heed her, to lie with her or to be with her. But it happened about this time, when Joseph went into the house to do his work, and none of the men of the house was inside, that she caught him by his garment, saying, “Lie with me.” But he left his garment in her hand, and fled and ran outside. (Genesis 39:7-12).
For the commandments, “You shall not commit adultery,” “You shall not murder,” “You shall not steal,” “You shall not bear false witness,” “You shall not covet,” and if there is any other commandment, are all summed up in this saying, namely, “You shall love your neighbor as yourself”(Romans 13:9).
An all-time favorite re-post... [Words of Comfort -- Ray Comfort's Blog] 2012-08-24 09:34
http://bit.ly/howtobecomeanatheist
Have a good weekend.
Feed the Mind. [Words of Comfort -- Ray Comfort's Blog] 2012-08-23 09:30
Hitchens & Twain [Words of Comfort -- Ray Comfort's Blog] 2012-08-22 09:30
The Sleeping Conscience [Words of Comfort -- Ray Comfort's Blog] 2012-08-21 09:30
Fearing Heaven [Words of Comfort -- Ray Comfort's Blog] 2012-08-20 09:30
“I believe that when I die I shall rot, and nothing of my ego will survive. I am not young and I love life. But I should scorn to shiver with terror at the thought of annihilation. Happiness is nonetheless true happiness because it must come to an end, nor do thought and love lose their value because they are not everlasting. Many a man has borne himself proudly on the scaffold; surely the same pride should teach us to think truly about man's place in the world. Even if the open windows of science at first make us shiver after the cozy indoor warmth of traditional humanizing myths, in the end the fresh air brings vigor, and the great spaces have a splendor of their own.”
“I don't believe in an afterlife, so I don't have to spend my whole life fearing hell, or fearing heaven even more. For whatever the tortures of hell, I think the boredom of heaven would be even worse.”
Life’s Transient Pleasures [Words of Comfort -- Ray Comfort's Blog] 2012-08-17 09:30
“For the creation was subjected to futility, not willingly, but because of Him who subjected it in hope; because the creation itself also will be delivered from the bondage of corruption into the glorious liberty of the children of God. For we know that the whole creation groans and labors with birth pangs together until now. Not only that, but we also who have the firstfruits of the Spirit, even we ourselves groan within ourselves, eagerly waiting for the adoption, the redemption of our body” (Romans 8:20-23).
The Uncomfortable Yoke [Words of Comfort -- Ray Comfort's Blog] 2012-08-16 09:30
“For to be carnally minded is death, but to be spiritually minded is life and peace. Because the carnal mind is enmity against God; for it is not subject to the law of God, nor indeed can be” (Romans 8:6-7).
Becoming Dumber [Words of Comfort -- Ray Comfort's Blog] 2012-08-15 09:30
Ten years ago, this would never have happened. Then, there was no doubt that "before the big bang" made no sense. But today, the certainty has gone. There is no escaping the inconvenient truth that Hubble's graph, work of genius though it is, contains a huge problem. It tells us that everything we see in the universe today -- us, trees, galaxies, zebras, emerged in an instant from nothing. And that's a problem. It's all effect, and no cause. The idea of "everything from nothing" is something that has occupied physicist Michio Kaku for much of his professional life.
You know, the idea sounds impossible. Preposterous. I mean, think about it -- everything from nothing! The galaxy, the stars in the heavens coming from a pinpoint. I mean how can it be? How can it be that everything comes from nothing? But you know, if you think about it a while, it all depends on how you define "nothing".
In Sandusky, Ohio, is Plum Brook Station. It is here that NASA recreates the conditions of space on Earth, and part of that means generating nothing. ..in vast quantities. This is the biggest vacuum chamber in the world. Its eight-feet-thick walls are made from 2,000 tons of solid aluminum. It takes two days of pumping out the air, and another week of freezing out the remaining molecules to create a near-perfect vacuum. A cathedral-sized volume of nothing. When they switch this place on, this is as close as we can get to a state of nothingness. Everywhere we look we see something. We see atoms, we see trees, we see forests, we see water. But hey, right here, we can pump all the atoms out, and this is probably the arena out of which genesis took place. So if you really understand the state of nothing, you understand everything about the origin of the universe.
Except, of course, it isn't quite that straightforward. For a start, the "nothing" created by NASA still has dimensions -- this is nothing in 3-D. And the tests carried out within the chamber can, of course, be viewed. This is nothing through which light can travel. NASA's "nothing" has properties. This "nothing" is, in fact, something.
“When physicists say there was ‘nothing’ before the Big Bang of our universe they do not mean literally “nothing.” What they mean is that there was no matter, just energy.”[2]
Atheism’s Illegitimate Father [Words of Comfort -- Ray Comfort's Blog] 2012-08-14 09:30
The Bible is like a fiddle [Words of Comfort -- Ray Comfort's Blog] 2012-08-13 09:30
Now a certain ruler asked Him, saying, “Good Teacher, what shall I do to inherit eternal life?” So Jesus said to him, “Why do you call Me good? No one is good but One, that is, God. You know the commandments: ‘Do not commit adultery,’ ‘Do not murder,’ ‘Do not steal,’ ‘Do not bear false witness,’ ‘Honor your father and your mother.’ ” And he said, “All these things I have kept from my youth.” So when Jesus heard these things, He said to him, “You still lack one thing. Sell all that you have and distribute to the poor, and you will have treasure in heaven; and come, follow Me” (Luke 18:18-22).
“Atheism by itself is of course not a moral decision or a political one of any kind. It simply is the refusal to believe in a Supernatural dimension. For you to say of Nazism, that it was the implementation of the work of Charles Darwin is a filthy slander, undeserving of you and an insult to this audience. Darwin’s thought was not taught in Germany. Darwinism wasn’t derided in Germany along with every other form of unbelief. All the great modern atheist thinkers: Darwin, Einstein and Freud were alike despised by the National Socialist Regime.”[1]
Atheist blogs [Words of Comfort -- Ray Comfort's Blog] 2012-08-10 09:30
Sinful nature. [Words of Comfort -- Ray Comfort's Blog] 2012-08-09 09:30

Every one of us is born with a sinful nature that has a propensity to sin—human beings have a natural bend toward things that God tells us are morally wrong: fornication, lust, greed, selfishness, adultery, lying, stealing, blasphemy, hatred, murder, abortion, rape, homosexuality, and some are even attracted to pedophilia. There are many different temptations that work in concert with our sinful nature, and sometimes people choose to follow a certain course. The supposed “gay” gene occasionally referred to, is just our inherited sinful nature. We are born with it.
If a pedophile takes pictures of naked children through a two-way mirror and can prove to you that no child is harmed by the practice—that they are in fact benefitted by it because their parents are well-paid, would you fight for the pedophile’s rights? If you would, then please send me your name and address. The local police in your area would like to alert your neighbors that you live nearby.
The Quiet Sound of Evidence [Words of Comfort -- Ray Comfort's Blog] 2012-08-08 09:30

We often have a studio audience when broadcasting "On the Box" -- our daily
webcast. One day there were only two people in the audience-a father (Larry) and his son. Larry had been a fisherman in his youth and had tragically lost one arm in machinery while on his boat. He had a great sense of humor and told us how having only one arm often came in handy when sharing his faith.
At the end of the broadcast, I did what I often do to give a sense of closure to the program. I held up a sign that told the studio audience to applaud. Larry's son didn't applaud much at all. He was too busy looking at his one-arm dad trying to clap with one hand. The program didn't have much closure that day. It did, however, remind me that the sound of that one-arm man clapping produces is the same sound that evolution believers produce when asked for sound scientific evidence for their theory.
Public domain picture.
Checking Out, and the Beatles [Words of Comfort -- Ray Comfort's Blog] 2012-08-07 12:05
Caught Lying about Stealing [Words of Comfort -- Ray Comfort's Blog] 2012-08-06 09:30
Strange Fire Conference #5: Conrad Mbewe [Pyromaniacs] 2013-10-31 11:21
by Dan Phillips
First post
Second post
My overall summary report to CBC
Third post
Fourth post
I am going to combine Conrad Mbewe's two sessions, on days one and three, into two posts.
Mbewe (mm-BAY-way) pastors Kabwata Reformed Baptist Church in Lusaka, Zambia, and is known as "the Spurgeon of Africa," though he himself does not know who gave him that name. When Phil Johnson introduced Mbewe's second talk, he said that, if he were unable to sit under John MacArthur's ministry, he'd gladly sit under Mbewe's preaching.
In his first talk, Mbewe focused on the impact of the spread of Charismaticism to Africa. He began with John 17:17 and noted that, were that verse fully valued, Charismaticism would never have taken hold. The central problem is a failure to recognize the centrality and sufficiency of the Word of God. Amen, and not just in Africa.
We all know that the dark ages are upon us again here in Africa. It is almost like a dark blanket that is slowly surrounding the land. People who know absolutely nothing of the core values of evangelical Christianity—the new birth, repentance and saving faith, justification and holiness, etc.—have hijacked evangelical Christianity in Africa. Even the term "born again" is being peddled without an iota of the meaning that Jesus had in mind when he used the phrase in his talk with Nicodemus. These are dark days indeed.Not only in Africa, sad to say. And the deafening squeal of protests to this conference, from perpetrators and enablers alike, signals that the darkness mightn't be ending anytime soon, due in large measure to the very tragic phenomenon we see in Jeremiah 5:31a:

Strange Fire Conference #4: Steve Lawson on Calvin and the Charismatics [Pyromaniacs] 2013-10-30 09:38
by Dan Phillips
First post
Second post
My overall summary report to CBC
Third post
Steve Lawson's session focused on what John Calvin would say to modern Charismatics. Lawson is a lot of things: senior pastor of Christ Fellowship Baptist Church in Mobile, Alabama, teaching fellow with Ligonier Ministries, visiting professor at the Ligonier Academy, The Master’s Seminary, and Samara Theological Seminary in Russia — and a constant fixture at conferences. (Seriously: how do you get that gig?)
It was really a terrific session. Lawson showed a surprising facility for spontaneous humor, particularly on display when a remark drew one person's applause ("Thank you to the one person who clapped" — and then, when everyone applauded, "I can tell when you don't mean it").
Having read Lawson's recent book on Luther (the review of which I plan to post after this series), I appreciate all the more the extent of research that goes into his talks. This was laced liberally with direct quotations from Calvin.
Lawson observed that Calvin faced foes who also believed they had inner light and direct revelations from God — the Anabaptists and the Libertines. He quotes Calvin as saying that they were 100 times worse than Roman Catholicism. Some of the libertines wore torn robes and seemed to want a "grunge" look. (When the audience laughed, Lawson remarked, "I feel like I just stepped on something," to more laughter.)
Lawson largely found Calvin's thoughts revealed in his commentaries on related passages. I looked them up to add to my notes and to BibleWorks as needed. Here are just some of the highlights:
On Acts 2:38, Calvin said:
In demanding miracles from us, they act dishonestly; for we have not coined some new gospel, but retain the very one the truth of which is confirmed by all the miracles which Christ and the apostles ever wrought.In other words, Calvin saw the purpose of the gifts as being to confirm the new message of the gospel and its messengers. When his contemporary critics demanded that he and the other reformers perform miracles, his response in effect was "Why should we? We are not bringing any new message, but the old message which has already been revealed and attested by God's supernatural power."
Lawson also noted that, when challenged as to why he invested so much time in responding to errorists, Calvin replied, "Even a dog barks when he sees someone assault his master." This clearly resonated deeply with us who heard. This foolish error opens the door to Satan, leads the simple away from the truth, and provokes God. For Calvin, a charismatic Calvinist would be an oxymoron.Prophecies had now almost ceased many years among the Jews, to the end they might be more attentive and desirous to hear the new voice of the gospel. Therefore, seeing that prophesying, which was in a manner quite ceased, doth now after long time return again, it was a token of a more perfect state. Notwithstanding, it seemeth that the same was the reason why it ceased shortly after; for God did support the old people with divers foretellings, until Christ should make an end of all prophecies. Therefore, it was meet that the new kingdom of Christ should be thus furnished and beautified with this furniture, that all men might know that that promised visitation of the Lord was present; and it was also expedient that it should last but for a short time, lest the faithful should always wait for some farther thing, or lest that curious wits might have occasion given to seek or invent some new thing ever now and then. For we know that when that ability and skill was taken away, there were, notwithstanding, many brain-sick fellows, who did boast that they were prophets; and also it may be that the frowardness of men did deprive the Church of this gift. But that one cause ought to be sufficient, in that God, by taking away prophecies, did testify that the end and perfection was present in Christ; and it is uncertain how these maids did execute the office of prophesying, saving that the Spirit of God did so guide and govern them, that he did not overthrow the order which he himself set down. And forasmuch as he doth not suffer women to bear any public office in the Church, it is to be thought that they did prophesy at home, or in some private place, without the common assembly.
Update: On the Record [Pyromaniacs] 2013-10-30 05:36
Strange Fire Conference #3: Joni Eareckson Tada and R. C. Sproul [Pyromaniacs] 2013-10-29 11:09
by Dan Phillips
First post
Second post
My overall summary report to CBC
The next session featured Joni Eareckson Tada. I expect you know her story: at age seventeen, Joni's dive into unexpectedly shallow water resulted in her becoming a quadriplegic for life. Her first book Joni, written in 1976, is a gut-wrenching read. Here faithful yet vulnerably candid style has remained her trademark. Joni's talks are chats rather than addresses; they are personal testimonies mixed with spontaneous singing of snatches of hymns. I find it impossible to listen to her without being moved, and without coming away thinking, "Yeah, I don't really have problems."
This was a very touching, personal session. Joni and MacArthur have known each other for a long time, and a lot of kidding has gone back and forth (she said that, when she decided to wear her wedding dress outside her chair, while other said she floated like an angel, Mac said she looked like a float). So she leaned on that friendship to call him up to the stage with her to sing an unrehearsed duet. It was very nice, and left few dry eyes.
Nor did Joni's occasionally tearful testimony concerning the path she's traveled. In early days, Charismatic friends kept wanting to command Joni's healing. Once, Joni was taken to a Kathryn Kuhlman meeting. They went hours early, to get front and center seating. However, Joni and the other folks in wheelchairs were hustled off to the side out of the way, where the spotlight never found them. Afterwards the ushers moved them out, a very quiet line of 35 souls, all untouched, unhealed, left to wonder why the Savior had in fact passed them by.
Joni shared the struggle she's had as her affliction has forced her to confront the corruption in her own heart. She has had cancer, she has severe chronic pain — just think about that for a moment — she has constant daily struggles. But in it, Joni has found a hope to hang on to that is very different than the focus of most prominent Charismatic leaders: Jesus, the Gospel, heaven. Joni shared that she and her husband Ken talk about pain being splashovers of Hell. Then what are splashovers of Heaven? Not happy days, they concluded, but finding Jesus in the splashovers of Hell.
In fact, one of the most poignant reflections Joni shared (in my words) was that a new, healthy body is not what she is most looking forward to about resurrection life. What she most looks forward to is the full healing of her heart, the final removal of its remaining corruptions and temptations. In other talks she has said that she plans to thank Jesus for her wheelchair, and for what He has taught her through it. It calls to mind Solzhenitsyn's "Bless you, prison, for being my life."
She noted that Jesus was never "about" healing; alluding to Mark 1, she noted that when healings threatened to take focus away from His preaching, He would move on.
As if Joni weren't dealing with enough pains and suffering, after the talk we were told that she'd had to leave right away because (as I understood it) she had some bleeding that they could not stop.
I spoke with friends on-site who were monitoring Twitter. They told me that, previous to Joni taking the stage, the carpers and critics were very active and strident. But when Joni spoke, they shut up. Ditto during talks by Justin Peters, who suffers from cerebral palsy.
The critics didn't (and don't) have much to say to folks like Joni and Justin. Lower back-pain, headaches, poor sense of smell? They're all over that. Sometimes they are, that is. Quadriplegia and cerebral palsy? Not so much. Off to the side of the stage, please.
This is "continuationism" in a nutshell, isn't it? One hundred years of desperately trying to prop up their position by argument, redefinition, and distraction — but in all the whole world not one faith healer commanding one Joni Eareckson Tada to "rise and walk," to any good effect. Kathryn Kuhlman's ushers bear mute testimony to the fact that they expect no such thing to happen. Why not? Because that gift, the ability to heal on command, has not in fact continued. Tens of thousands of smart phones have failed to capture one such occurrence, though they could have captured many in the days of Jesus and the apostles (cf. Mark 6:56). The very fact that they are forced to make verbal arguments for continuationism — rather than pointing to 1900 years of Joni's and Justin's hopping up out of their wheelchairs — is potent proof that their position is bankrupt, and that in their hearts they know it.
Then John MacArthur made a remark in passing that stuck with me. He pointed that in the whole sweep of Biblical history, there were not that many healings. This is particularly true in the Old Testament, where the miracles usually "ended up with a lot of people dead. Once, the whole world." I'd never seen it that way, but of course he's right. Another point of discontinuity between the genuine and the imitation — Charismatic leader Benny Hinn and his "Holy Ghost machine gun" to the contrary notwithstanding.
The next session was a video from R. C. Sproul, who was prevented from coming by ill health. Sproul briefly traced the history of the Pentecostal movement. It was an early-20th-century invention that had no traction into the mainstream until the middle of that century, after which it spread to the Roman Catholic church and elsewhere indiscriminately.
Sproul then made the point that the Spirit's coming in Pentecost has to be set in the context of the history of redemption. It isn't a "and then that happened" event. Sproul set up by contrast with Moses wishing that all the Lord's people could have the Spirit (Num. 11:29). What had been a wish on Moses' lips becomes a prophecy in Joel 2, and then a reality in Acts 2.
What we see in Acts is the Spirit coming to four people-groups: first the Jews in Jerusalem (Acts 2), then the half-Jew Samaritans (Acts 8), then the Gentile proselytes in Acts 11, and then the full-on Gentile converts in Acs 19. Each group receives its own reenactment of Pentecost, presided over by apostles, signifying their full reception of the Spirit. Unlike the false teaching of Pentecostals, in each case, every believer present received the Spirit.
I have often noted (and argued at some length in my unpublished book on the Holy Spirit) that trying to re-do this period in order to receive the Spirit is like trying to build a manger and gather some shepherds and angels to receive Christ. The manger is how Christ came into the world, and the events in Acts are how the Spirit formed the church. It isn't intended that we reproduce the historical events.
And so Sproul's argument was that the problem with Pentecostals is that they make too little of Pentecost, not too much; and that their understanding of it differs from the apostles'.
No profit from the prophet? [Pyromaniacs] 2013-10-27 03:01
Your weekly dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from The Gospel of the Kingdom, page 80, Pilgrim Publications."There is no suiting some people. Even the great Lord of all finds His wise arrangements met with discontent."
Many Ways to Read Scripture. Choose Wisely... [Pyromaniacs] 2013-10-25 03:01
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.Strange Fire conference: my report to our church [Pyromaniacs] 2013-10-24 09:30
by Dan Phillips
I continue to prepare more written reports and reflections on various sessions. Some have expressed interest in hearing the (60-minute!) summary I gave our church. Since we're here to serve, we recorded it; here it is:

Strange Fire Conference #2: Session 1, John MacArthur [Pyromaniacs] 2013-10-23 11:25
by Dan Phillips
Introduction. My intention in beginning these posts on sessions at the Strange Fire conference held last week at Grace Community Church will not be to reproduce everything that was said or done. You can consult such stalwarts who were there as Mike Riccardi, and eventually access the sessions themselves. I'll present highlights, impressions, conclusions that stood out to me.
Beyond this note, I won't comment on the singing that introduced each session, or special musical performances by GCC's soloists or the Master's Chorale — all of which were wonderful (particularly the latter).
John MacArthur fittingly welcomed us all, sharing that the Charismatic movement has been a concern of his since the first days of his ministry. He saw it as a threat, and wrote books on the subject in the late 1970s, the early 90s, and this month. This is the first conference he has ever held on the subject.
Are people saved within the Charismatic movement? Yes; but when they are, it is because of the gospel, which was not invented by that movement. God has always protected His gospel and raised up those who proclaim it, and He does so now. Some within Charismaticism also love and preach the Gospel, yet are heterodox (not heretical) when it comes to the Spirit.Anyone who has set aside the law of Moses dies without mercy on the evidence of two or three witnesses. 29 How much worse punishment, do you think, will be deserved by the one who has trampled underfoot the Son of God, and has profaned the blood of the covenant by which he was sanctified, and has outraged the Spirit of grace? 30 For we know him who said, “Vengeance is mine; I will repay.” And again, “The Lord will judge his people.” 31 It is a fearful thing to fall into the hands of the living God. (Hebrews 10:28–31)MacArthur noted that many groups of Christians have assembled to oppose the trampling underfoot of the Son of God by defending sound Biblical Christology, the first item mentioned by the writer. I could add that organizations like T4G and TGC and many others at least formally oppose the denigration of the Gospel and the blood of the covenant (the second item), by defending the Biblical Gospel.
Instead, what one hears (me talking now, not MacArthur) is wails and squeals about MacArthur talking about these abuses — not about the abuses themselves. It reminds me of how shocked (shocked!) Senatrix Barbara Boxer was for Senator Rick Santorum to describe partial-birth abortion on the floor of the Senate. The procedure itself didn't bother her a bit, she adores it as a sacred right. But describing it? Offensive! Unheard-of!
Strange Fire Conference #1: personal [Pyromaniacs] 2013-10-23 07:45
by Dan Phillips
Here begins a series of posts on the Strange Fire conference held last week at Grace Community Church.
This may be the briefest, as it will be personal. I find people are often more interested in posts like this than I would ever in my most febrile moments imagine, so here 'tis. If you're not one of those people, sorry, really I am; see you next time.
Big Gulp. Dear wife and I flew out Monday evening, leaving our boys (14 and 18) alone for the first extended time. With a brilliance that I display only when I don't try, I had just preached a sermon on God's surprising cure for anxiety, which I selected without thinking how much I'd need it myself. We were helped in knowing that many in our dear church were available to them and would check up on them, perhaps performing unannounced inspections and the like.
Flight. But we ran into a (wait for it) glitch right away. Frontier Airlines, whose reputation for frequent cancellations and re-routings seems to be well-earned, wanted to re-route us due to a "routine maintenance." We wondered why, if it were routine, it caught them by surprise. But never mind; initially it looked like a windfall, in that we'd get a direct flight to LA instead of having a layover in Denver. Win, right? We only lightly detected the ominous overtones in the agent's promise, "I'm going to try really hard to get your luggage transferred to your new flight."
Heroic though her efforts may have been, they were not successful. We had a fairly uncomfortable ride in the very back two seats, hard against the bathroom, next to the only surly and unfriendly flight attendant I've ever encountered. When we landed — no luggage. Not a total surprise, so we logged our case with the agent after a long wait, and went to our room. Valerie had found a lovely Comfort Inn motel near Universal Studios, with a very nice, decent-priced room, great breakfasts, and best of all, about equidistant from Grace Community Church and Bob's Big Boy.
To close those loops:
At Grace itself, it was great seeing Travis Allen (who has a formidable memory) and Jay Flowers again. One of the highlights for Valerie and me was enjoying dinner with m'man Mike Riccardi, who's flourishing at GCC and Master's, and busier than a one-eyed cat watching six mouse-holes. Also got to renew acquaintances briefly with Phil's son Jeremiah Johnson and meet his lady-friend. Jeremiah was among those enjoying Valerie's life-changing peanut-brittle ministry when we visited his parents a couple of years back.
Spurgeon the Cessationist [Pyromaniacs] 2013-10-20 03:01
Your Strange Fire Dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from The Metropolitan Tabernacle Pulpit, volume 58, sermon number 3,290 "God's hand at eventide.""Faith healing is grand, but faith-enduring is grander."
What Lutherans have done for 5 Centuries [Pyromaniacs] 2013-10-20 23:19
A Dangerous Doctrine Calls For a Candid Response [Pyromaniacs] 2013-10-18 03:01
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.On The Record [Pyromaniacs] 2013-10-22 16:37
The danger of denial [Pyromaniacs] 2013-10-13 03:01
Your weekly dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from The Gospel of the Kingdom, page 74, Pilgrim Publications."Whosoever therefore shall confess me before men, him will I confess also before my Father which is in heaven. But whosoever shall deny me before men, him will I also deny before my Father which is in heaven."
Who is YOUR pastor? No, really... [Pyromaniacs] 2013-10-11 03:01
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.We ask you, brothers, to respect those who labor among you and are over you in the Lord and admonish you, and to esteem them very highly in love because of their work. Be at peace among yourselves (1 Thessalonians 5:12-13).
Obey your leaders and submit to them, for they are keeping watch over your souls, as those who will have to give an account. Let them do this with joy and not with groaning, for that would be of no advantage to you (Hebrews 13:17).
Fascinating people and reading narrative portions of Scripture [Pyromaniacs] 2013-10-09 09:37
by Dan Phillips
As I read through Acts in the morning, I'm struck anew by what fascinating people Luke introduces — and how little he tells us of them.
Take Simon Magus in chapter 8. Wouldn't you like to know a lot more about him, from Luke? When Luke says he used to do magic, what does that mean? Actual magic, or tricks? What kind? And what was his background? Also, what did he do in response to the apostles' rebuke? He figures prominently in post-NT writings, but is any of that accurate? What became of him?
Or the Ethiopian eunuch in that same chapter. What an interesting fellow this man is! We read his story anachronistically, and just think how cool it is that he was in Isaiah 53 instead of Leviticus 21:20. But what about the fact that he had a copy of Scripture at all? How rare was that? How did he come by it, how wealthy must he have been! Or was the scroll Candace's? What became of him? The same Holy Spirit who told Philip to go preach to him also told Philip (in effect) not to do any followup with him. So the eunuch went his way rejoicing, we read... and then what? Wouldn't you love to know?
Or Lydia the business lady in chapter 16, or the demon-possessed slave girl in the same chapter — what different women, yet side by side in the narrative. What was their background, and what came of them after these encounters?
These and many other figures crowd the book of Acts alone, to say nothing of the other Biblical books.
But what we as readers (and particularly as preachers) must remind ourselves is that the text of Scripture is what is God-breathed and profitable, and that must always be our focus. It isn't the stories that hold this place, nor the people. It is the text, the words of Scripture, that must be our focus.
It is the text that reflects and unfolds the mind of God. In that text, God told us everything He wants to know, which means He told us everything we need to know. So we must both discipline ourselves not to run off on rabbit-trails, and to focus on what's there to see the mind of God. What we want to know about Simon, the eunuch, Lydia and the rest is not the same as what God wants us to know — and we must see to it that we focus on the latter, not the former.
In fact, this is all the more arresting and important in proportion to how fascinating the person is. God in effect is saying to us "Never mind all that, don't let the shiny objects distract you: this is what I want to impress upon you."
So it doesn't matter what kinds of snakes bit the Israelites in Numbers 21, or how big the copper serpent was, or how long the pole, or any of that. What matters is that those snakebit Israelites who believed Yahweh's words and looked, lived — and no one else. And on and on.
We know that the main thing is the main thing; we need to remember that the text of Scripture is always the main thing. If it were important, God would have told us. If He told us, it is important.
Reviviscence [Pyromaniacs] 2013-10-06 03:01
Your weekly dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from The Sword and the Trowel, March, 1898, Pilgrim Publications.If you know anything of the work of God’s grace in your own heart, you will frequently have to pray, “O Lord, revive Thy work.”
"God...maketh use of means" (WCF) [Pyromaniacs] 2013-10-04 03:01
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.Written-by-man dodge (NEXT! #38) [Pyromaniacs] 2013-10-01 03:01
by Dan Phillips

"Filled" [Pyromaniacs] 2013-09-29 03:01
Your weekly dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from The Gospel of the Kingdom, Pilgrim Publications, page 22."Blessed are they which do hunger and thirst after righteousness: for they shall be filled."
"A meditation on Psalm 90" [Pyromaniacs] 2013-09-27 09:18
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.Pyro Brain Trust question: missionary recommendations? [Pyromaniacs] 2013-09-24 08:54
by Dan Phillips
Foreign missions, per se, is a topic we haven't treated with any frequency here. My purpose in bringing it up now is brief and specific. It's to ask this:
What missions do you personally know (A) that specifically target unreached people or Muslims, and (B) that do so with pure Gospel preaching (preferably Calvinistic, cessationistic, baptistic), and (C) that do so with any effectiveness?

What can't be cured must be endured [Pyromaniacs] 2013-09-22 03:01
Your weekly dose of Spurgeon
The PyroManiacs devote some space each weekend to highlights from the lifetime of works from the Prince of Preachers, Charles Haddon Spurgeon. The following excerpt is from Lectures to my students, "The blind eye and the deaf ear," Pilgrim Publications, pages 167-68."What can’t be cured must be endured, and the best way of enduring it is not to listen to it."
Neither disdain nor slavish acceptance [Pyromaniacs] 2013-09-20 03:01
From 2006 to 2012, PyroManiacs turned out almost-daily updates from the Post-Evangelical wasteland -- usually to the fear and loathing of more-polite and more-irenic bloggers and readers. The results lurk in the archives of this blog in spite of the hope of many that Google will "accidentally" swallow these words and pictures whole.The Bible-only(-except-for-me!) dodge (NEXT! #37) [Pyromaniacs] 2013-09-18 06:57
by Dan Phillips

Response to Dr. Ally, Part III [Alpha and Omega Ministries] 2013-10-30 23:54
Before flying to Vancouver over the weekend I began responding to an article posted by Dr. Shabir Ally relating to the substance of our debate at the University of Pretoria in South Africa, which took place on Tuesday, October 8th. I had come to the fourth point in Dr. Ally’s presentation:
My fourth reason for thinking that the original disciples did not consider Jesus God is that Paul’s writings bear evidence that he was in conflict with the original disciples not only over questions of law but also over the question of monotheism. In 2 Corinthians 11:4, it is clear that Paul’s opponents were preaching what Paul calls ‘another Jesus.’ Elsewhere in Paul’s writings it becomes clear that his opponents are the original disciples of Jesus and close followers of the disciples. Now, as Bruce Chilton mentioned, the original disciples’ response to Paul’s accusations are not found in the New Testament. Given the chance, the disciples can be expected to say that their Jesus was the original Jesus, and Paul’s Jesus was the ‘other Jesus.’
I wanted to spend a little time on this claim because it will probably not make much sense to most in our audience. Christians who hear sound, consistent, believing teaching will have no basis upon which to even understand the allegation being made. So I wanted to make sure it was clear so that its refutation can be understood as well.
Today it is very popular in skeptical scholarship to begin with the most egregiously indefensible presuppositions and then force them upon the text. Clearly, the scholars Dr. Ally looks to begin not with the assumption of the unity of the New Testament, but its disunity. They feel free to theorize about “earlier forms” of books, cut and edit as they see fit, introduce contradiction, etc. Nowhere is this seen with more clarity than in this particular assertion which has become very popular today, at least amongst the radical skeptics.
What lies behind Dr. Ally’s argument is the idea that there was a massive rift between Paul and the other Apostles, such as Peter. Note his words, “Elsewhere in Paul’s writings it becomes clear that his opponents are the original disciples of Jesus.” It is being argued today that the people Paul referred to as the “super Apostles” were, in fact, Peter and James and the rest of the Jerusalem leadership, and that there was a great schism between Paul and these original disciples of Jesus. So, then, Dr. Ally hopes to insert a rift into the text of the Bible, turning Paul into an opposer of the “true disciples of Jesus.”
How should we respond to such an assertion? Well, first, let’s address the text cited, that being 2 Corinthians 11. The primary interpretive issue is the identity of the τῶν ὑπερλίαν ἀποστόλων, “the foremost” or “super” apostles (depending on how we understand the phrase to be used here). Two primary possibilities exist, as laid out in the following citations from recognized commentators:
5. The superlative apostles (Gk hyperlian apostoloi) to whom, according to Paul’s opponents, he himself was so inferior ( cf. 12.11), can scarcely be other than the Jerusalem apostles, including James (as in Gal. 1.19). Such language, by whomsoever used, could not well be applied to men of lower apostolic status than theirs. By this time, perhaps, none of the Twelve was actually resident in Jerusalem, but Jerusalem would still be regarded as their home base. Their designation as superlative apostles might conceivably go back to the intruders in Corinth, who by this phrase, invoked the authority of men whose commission and status were so incomparably superior, by their account, to anything that Paul could justly claim; but there is a strong flavour of irony about the expression, and it is more likely that it is Paul’s way of summing up his opponents’ portrayal of the Jerusalem leaders. We may compare his reference in Gal. 2.9 to ‘James and Cephas and John, who were reputed to be pillars’; it may indeed be these three who are primarily in view here. Paul has no thought of depreciating their apostolic status; he is at pains to emphasize that his is not in the least inferior to theirs. He had received his commission from the risen Christ (I C. 9.1; 15.8; Gal. 1.12), and, by his own account, so had they (1 C. 15.5-7). Even if he permits himself a measure of irony, it is rather at the expense of his opponents’ portrayal of the Jerusalem apostles than at the expense of the apostles themselves; in fact, whatever he may have thought or felt about the failure to observe the delimitation of mission fields agreed upon at Jerusalem, he is studiously careful to avoid any overt criticism of the Jerusalem apostles, while he is unsparing in his denunciation of the intruders who invoked their authority ( cf. verses 13-I 5). F.F. Bruce, The New Century Bible Commentary, I&II Corinthians, (Eerdmans, 1971, pp. 236-237)
Paul continues to use the first person singular (see vv. 1, 2, 3) and states his own opinion about the infiltrators. He compares himself with them and facetiously calls them superapostles. He repeats this name in the next chapter, where he again states that he is not inferior to these people (12:11; see also 11:23). By resorting to derision, Paul implicitly indicates that the Corinthians already should have evaluated the intruders as impostors. Indeed, they needed to come to Paul’s defense and dismiss his rivals.
Who are these so-called superapostles? Are they Jesus’ twelve disciples and others who followed him from the time of his baptism to that of his ascension (Acts 1 :21-22)? This interpretation fails to do justice to the immediate context, in which Paul speaks of an opponent who preaches a different Jesus (see v. 4). Moreover, the three pillars of the church (Peter, James, and John) had come to an agreement with Paul on a division of labors between Peter and Paul (Gal. 2:6-9). Apart from a confrontation at Antioch, we do not read of any tension between these two apostles (Gal. 2:11-14) or the rest of them. Hence, we cannot infer that Paul considers himself inferior to the Jerusalem apostles. Rather, he employs irony when he labels the Judaizing interlopers as superapostles.
The expression superapostles “even linguistically brings out the impossible nature of such apostles,” because being an apostle of Jesus is in itself incomparable. The list of spiritual gifts indicates no higher position than that of apostle (I Cor. 12:28; Eph. 4:11).
If the superapostles are not identified with the apostles in Jerusalem, we must associate them with the false apostles whom Paul mentions in verse 13. These men came to Corinth on their own accord, adopted the name apostles to gain entry into the church, and gave the impression of possessing more authority than Paul. Simon Kistemaker, New Testament Commentary, 2 Corinthians, (Baker, 1997), p. 365.
The sense here again depends on the connection. If the γὰρ refers to v. 4, the reference must be (as so often occurs in Paul’s writings) to a thought omitted. ‘Ye are wrong in thus bearing with the false teachers, for I am equal to the chief apostles.’ This, however, is not in harmony with the context. Paul’s design is not so much to reprove the Corinthians for tolerating the folly of the false teachers as to induce them to bear with his. He felt it to be necessary to vindicate himself, and he therefore prays them to bear with him a little in his folly. To this point every thing here refers. They should thus bear with him, I. Because he was jealous over them with a godly jealousy. 2. Because they would bear with any who really preached another gospel, were that possible. 3. Because he was on a par with the chief apostles. The connection, therefore, is not with v. 4, but with the main subject as presented in v. 1. This also determines the question, Who are meant by the chiefest apostles? If the connection is with v. 4, then the expression is to be understood ironically in reference to the false teachers. ‘Ye do wrong to tolerate them, for I am in no respect behind those superlative apostles.’ So Beza, Billroth, Olshausen, Meyer, and the majority of the moderns. The reason given for this is, that there is no controversy with the true apostles in this connection, and therefore nothing to call for such an assertion of his equality with them as we find in Gal. 2, 6-11. There is, however, no force in this reason if the connection is with v. I. ‘Bear with me in my boasting, for I am not behind the chiefest apostles.’ In this view the reference to the true apostles is pertinent and natural. Paul says, μηδὲν ὑστερηκέναι, that as to nothing, in no one respect, had he fallen short, or was he left behind by the chiefest apostles; neither in gifts, nor in labours, nor in success had any one of them been more highly favoured, nor more clearly authenticated as the messenger of Christ. He was therefore fully entitled to all the deference and obedience which were due to the chiefest apostles. The expression τῶν ὑpερλίαν ἀpοστόλων, is not in itself bitter or ironical. This is a force which must be given by the connection; it does not lie in the words themselves. It is not equivalent to the ψευδαpόστολοι of v. 13, and therefore there is no more reason why the true apostles should not be called οι ὑpερλίαν ἀpόστολοι than οἱ δοκοῦντες εἶναί τι in Gal. 2, 6. The argument, therefore, which the Reformers derived from this passage against the primacy of Peter is perfectly legitimate. Paul was Peter’s equal in every respect, and so far from being under his authority, he not only refused to follow his example but reproved him to his face. Gal. 2, 11. Charles Hodge, I&II Corinthians (1859) pp. 631-632.
He then proceeds to refute the two reasons which were assigned for the disparagement of his apostolic authority, viz., (a) he had none of the arts of a trained rhetorician, (b) he had not claimed maintenance from the Church of Corinth, which he had a right to do, if of genuine “apostolic” rank. οἱ ὑπερλίαν ἀπόστολοι, “these superfine Apostles” is thus, as at 12:11, an ironical description of the ψευδαπόστολοι (ver. 13) against whom he is contending. The A.V. and R.V. render “the very chiefest Apostles,” i.e., the original Twelve, who received their commission directly from Christ, and especially Peter, James and John; but to introduce any mention of them here would be irrelevant, and would interrupt the argument (they were ἰδιῶται ἐν λόγῳ), not to speak of the fact that ὑπερλίαν seems always in Greek literature to be used in an ironical sense. Benard, J. H. (n.d.). The Second Epistle of Paul to the Corinthians. The Expositor’s Greek Testament: Commentary. New York: George H. Doran Company.
1. If οἱ ὑπερλίαν ἀπόστολοι are (οἱ) ψευδαπόστολοι (11:13), that is, Paul’s rivals at Corinth, then the expression “the superlative apostles” is either the self-description of “the false apostles” or the Corinthians’ appraisal of “the false apostles” or Paul’s sarcastic/ironical description of “the false apostles” or of their own opinion of themselves.
2. If οἱ ὑπερλίαν ἀπόστολοι are not (οἱ) ψευδαπόστολοι, then the expression “the superlative apostles” is either “the false apostles’ ” description of the Twelve or the so-called three “pillars” (στῦλοι, Gal. 2:9, namely James, Cephas, and John) in the church of Jerusalem or Paul’s own commendatory or derogatory description of the Twelve or the Three or his parodying of the Corinthian view of “the false apostles” or his own ironical description of the exalted view of the Twelve held by “the false apostles.”
In the Introduction I have endeavored to defend this last position. The most compelling arguments in favor of drawing a distinction between “the superlative apostles” and “the false apostles” (#2 above) are these. First, it is difficult to imagine that Paul would refer to himself as “in no way inferior” to false teachers whom he describes as “deceitful workmen” (11:13) and servants of Satan (11:15). It would be very appropriate for him to claim equality with the Twelve or the Three, but wholly incongruous to claim to be not a whit behind “false apostles.” Second, when Paul compares himself with the “false apostles” he speaks boldly and positively and claims superiority (“… so am I [11:22, three times] … I am more … much harder … more frequently … more severely,” 11:23 [NIV]), but when he compares himself with “the superlative apostles” he speaks mildly and negatively and implies equality (“I am not at all inferior,” 11:5; 12:11). Third, the apostles who are “false” provoke Paul’s forthright and direct denunciation (11:13, 15), even if he takes their allegations and claims seriously, whereas he treats the apostles who are “superlative” indirectly (11:5; 12:11) and with a gentle irony that is comparable to his depiction of the Three as “those who were reputed (οἱ δοκοῦντες) to be pillars” (Gal. 2:9; cf. Gal. 2:6). Fourth, whatever the source of the expression οἱ ὑπερλίαν ἀπόστολοι, would Paul himself have applied the term ἀπόστολοι, however understood, to those he describes as ψευδαπόστολοι? This phrase, “by whomsoever used, could not well be applied to men of lower apostolic status than theirs,” namely “the Jerusalem apostles, including James” (Bruce 236–37).
In 10:12 Paul disavows comparison between himself and his rivals, although he engages in such in 11:22–29 as part of his κατὰ σάρκα boasting (11:18). Here in 11:5, on our view, he resorts to comparison between himself and the Jerusalem leaders, claiming that he is “in no respect” (μηδέν) inferior to them. μηδέν is surprising, since Paul was not a Judean Jew, was not a member of the mother church in Jerusalem, and was without a personal acquaintance with Jesus. But a rigorist understanding of μηδέν is inappropriate in the context. μηδὲν ὑστερηκέναι, “to be in no way inferior,” is litotes for εἶναι ἴσα ἐν παντί (cf. Phil. 2:6), “to be equal in every way,” and Paul has in mind his parity of status as an apostle (as in 1 Cor. 9:1; 15:5, 8–11) (note τῶν … ἀποστόλων) and as a person competent in knowledge of the faith (γνῶσις, 11:6). This assertion of equality is a response to unfavorable comparisons between Paul and the original apostles being made by his rivals who were illegitimately invoking the authority of the Twelve (and James) in support of their own Judaizing program at Corinth. Paul’s overall point in v. 5 is that if the Corinthians tolerated intruders who brought a counterfeit gospel (v. 4) and made inflated claims concerning the Jerusalem leadership (cf. v. 5), they ought also to bear with him in his “bit of foolishness” (v. 1). Murray J. Harris (2005). The Second Epistle to the Corinthians: a commentary on the Greek text (pp. 746–748). Grand Rapids, MI; Milton Keynes, UK: W.B. Eerdmans Pub. Co.; Paternoster Press.
Let me summarize. If οἱ ὑπερλίαν ἀπόστολοι refers to Peter, James, and John, it would be referring to the interloper’s use of their names as a means of denigrating Paul’s authority in the church in Corinth. It would not be a slight upon his fellow Apostles themselves. If the phrase does not refer to them (depending on whether you take v. 5 as being connected directly to v. 1 or v. 4) then it would be a direct reference to the interlopers and their pretended authority, paralleling v. 13. In either case, there is nothing in the context to even begin to insinuate that Paul is saying the original disciples of Jesus are preaching a different Jesus than he is preaching.
So how can modern hyper-skeptics come to the conclusion that there was this huge rift between Paul and Peter when the text nowhere even hints at such a thing? The starting assumption must be recognized. There is one assumption that must be made right at the start, though it is rarely stated as openly as it should be. The Apostle Paul was a liar. Simple, yes? There is no way to sugar-coat it. You have to start out with the simple belief that what Paul wrote in his epistles is dishonest, misleading, erroneous, and purposefully so. Paul was a liar. And Luke, to quote one recent popular writer (Reza Aslan), was Paul’s “sycophant,” so, he, too, is to be dismissed as a liar. So you begin with the assumption that nothing Paul or Luke says is true, and then you have an open door through which to drive a semi tractor worth of theories and speculations, unhindered by the actual historical documents you are pretending to interpret all along. Guilty until proven innocent, but, of course, there really is no mechanism allowed for proving Paul innocent since the starting point of the entire process is his own guilt!
Hence, if Paul says he and Peter agreed on the gospel—well, Paul is a liar. No evidence of a division between them about who Jesus was? Paul was a liar. Luke records the Acts 15 council and there is no division over who Jesus was or what He did? Luke was a liar, too. See how easy this is? Find evidence in the Petrine epistles that he taught what Paul taught? Peter didn’t write any of that anyway! So, once you say you don’t have anything that actually reflects Peter’s views, and Paul is a liar, and now you have the stuff of modern radical skepticism.
Of course, Dr. Ally would never, ever allow us to do this with Muhammad, or Abu Bakr, or Aisha, or any of the other early Muslim figures or writers. But he does so with Paul. In fact, this kind of anti-Paulinism is not only popular amongst liberal writers today, it is simply epidemic amongst Muslim apologists. It is easy to see why, for Dr. Ally admitted in our debate that Paul taught that Jesus was Yahweh in human flesh, and though Shabir still does not understand how that can be (he does not seem to comprehend the distinction between being and person, nor allow for such distinctions in his thought, though, again, he makes similar distinctions in his own theological concepts regarding Allah, Allah’s attributes, and the eternality of the Qur’an as uncreated), the fact remains that there is a fundamental, unalterable, and irreconcilable difference of thought and teaching between the epistles of Paul (and I would argue, all the New Testament writings) and the understanding of the Qur’an. Of course, I do not believe the author of the Qur’an was at all aware of this (if he was, why is there no warning against Paul? Why no refutation of Paul’s Christology and a vindication of Peter’s, for example?), but that is the problem: Dr. Ally’s view of the NT is forged by his understanding of a later work, a work that is fundamentally flawed in its understanding of the New Testament.
Dr. Ally’s fifth point he expressed this way:
Fifth, Jesus himself is known to have taught that he is a man and not God. But the Gospels distorted the image of Jesus transforming him from a man to something greater. This can be seen as we compare Mark, the first Gospel, to Matthew and Luke. But this evolution can be seen even more as we compare Mark with John, the last of the four Gospels to be written.
Briefly, once again, Shabir insists upon an anachronistic redefinition of Christian belief. Yes, Jesus taught He was a man. Well, He did not have to do that, everyone could see that. But Christians affirm the humanity of Jesus. Any orthodox confession speaks of this truth. But it is a presupposition that we have already challenged repeatedly that there is any such distortion of Jesus’ image, and repeating the same old “I can read the mind of Matthew and based upon my theories of gospel originations and assuming the gospel writers simply edited the earlier versions in a blind fashion I can come up with this theoretical conclusion” arguments will not do in this debate. The whole point of the debate was to get beyond the repetition of that old argument and ask the most basic question—is there any strata of the earliest tradition that is devoid of an exalted view of Jesus, and my answer was, no, there is not. And that is why the first part of this response was so important, because Dr. Ally conceded the point! By abandoning the New Testament evidence and going to the Old Testament Shabir was admitting the whole point of the thesis of the debate!
There is yet some more to respond to, which I hope to get to very quickly in the next portion of my reply.
Today on the Dividing Line: Steven Anderson, Strange Fire, David Allen, and More [Alpha and Omega Ministries] 2013-10-29 19:26
Covered a lot today, with a brief recounting of my interview with KJV Only advocate Steven Anderson, and then a longer series of comments based upon my listening to three of the presentations from the Strange Fire Conference on my ride this morning. Then we took calls, including one from Rory on the presentation of David Allen from Southwestern against the Reformed understanding of the atonement, specifically in regards to his attempt to make 1 Corinthians 15 a verbatim presentation by Paul to the Corinthians rather than (as the vast majority of scholars see it) a summary of the apostolic kerygma.
Here’s the audio of the program.
Here’s the video:
Today’s Sola Scriptura Conference LIVE FEED [Alpha and Omega Ministries] 2013-10-26 11:41
Is Homosexuality Compatible with Authentic Christianity? vs Barry Lynn is posted [Alpha and Omega Ministries] 2013-10-25 17:17
The Great Debate VI – Purgatory is posted [Alpha and Omega Ministries] 2013-10-25 17:15
Wilhelmus à Brakel on Sanctification and Holiness [Alpha and Omega Ministries] 2013-10-25 14:53
Wilhelmus à Brakel was one of the most influential men to come out of the Dutch Further Reformation. He wrote with a scholarly mind and a pastoral heart. He exemplified the maxim: Correct doctrinal thinking leads to godly living. His work, The Christian’s Reasonable Service is both challenging in its doctrine, and convicting in its piercing observations.
I had been reading his section on Sanctification and Holiness, when I read these very rich and poignant observations about the “old man”. I thought them worthy of posting. You can find an electronic version of all 4 volumes at this site.
The Functioning of the Old Man in the Believer
The old nature stirs up to the commission of sin.
(1) Sometimes it does so by violent assaults. The lusts are so agitated and are stirring so vehemently that there is no time to think upon the fear of God. Even if the fear of the Lord surfaces, the lusts increase so forcefully in strength that any good inclinations are immediately extinguished. Thus, sin is committed before one can think about anything else, the heart being carried about as chaff in the wind.
(2) Sometimes the old nature seeks some rest; to be so intently focused upon God tires the body and the mind, so that it appears impossible to live in such a manner. The old nature, in seeking some rest and relaxation, begins initially to think upon natural things; however, the lusts of the flesh begin to stir, and the thoughts pertaining to natural things become sinful, due to one‘s ego entering the picture. A person will begin to build castles in the sky, imagining himself to have possessions, to be in a position of prominence, of being honored, and of having riches. Even though he knows that he will never attain to this, he nevertheless entertains himself with such imagery. From this point the old nature proceeds to reflect upon that sin which most readily presents itself be it immorality, a lust for money, or pride. Being thus drawn away from his steadfastness, he commits sin to the degree that the moment permits, and if the opportunity were not lacking, he would fall into sins which he never thought himself to be capable of. Or, if the opportunity is there, he will fall into sin from which he thought to have been delivered be it in a natural sense or by grace.
(3) Sometimes the old nature gains in strength due to recklessness. A person will bring himself into situations, knowing from experience that they have repeatedly ensnared him. This can either be solitude, or the company of certain people, yet he is of the
opinion that he will now be able to abstain from the previous sins. In making use of the opportunity, however, he is inclined to it before realizing it, and sin having found an opening must proceed; the sin which is then at hand gains the upper hand. Contact with grease cannot but leave a stain.1
(4) Sometimes the old nature presents something as being beneficial but conceals its sinfulness. It presents it as a necessity, as being delightful, as being advantageous, or as being honest, etc. Sometimes it is presented as a white lie, as being a necessity
(not being able to do business otherwise), as being an honest deed, or as something which would otherwise prevent you from intermingling with people in a civil manner. Sometimes it suggests that one will thereby come into a position, in which he will be able to do more good subsequently and similar pretenses, which are not advanced in a premeditated manner, but suddenly present themselves at a given opportunity. And thus, man takes more liberty or at least he does not resist sin as much, and the old nature breaks through, one sin begetting another.Secondly, the old nature is likewise always engaged in keeping man from that which is good.
(1) There will be no time for one to engage in his godly exercises of praying, reading, singing, and meditation. Therefore these exercises either do not occur at all, or only in a casual manner to satisfy the conscience. It is as if he is rushed, even though he frequently would have the time.
(2) At another time one will postpone the matter, determining to do it, but to do it in a more quiet and composed manner; certain things first have to be accomplished. In the meanwhile time slips away or the Spirit has departed, and one does not get to it, or it is void of all spirituality.
(3) Then again the task appears as being exceptionally difficult; one looks up against it, and seeks to avoid and postpone it. Having burdened himself with many difficulties, he approaches the duty as a lazy person and, so to speak, crawls forward. It is too difficult and one is not fit to do it.
(4) Again he thinks that all that he does is in vain, that God does not hear, that one shall not obtain it, and he suggests to himself that he shall not obtain anything in the future anyhow. Our words do not carry any weight with others; we shall be put to shame, and our careful walk will only be construed as hypocrisy.
(5) Or one will try to compromise. The way to heaven is not so narrow as one generally claims. Would all those perish who are not so precise? No! It is not contrary to godliness to have determination, and to be courteous and cheerful. Thus, the old nature will prevent one from making vigorous progress and from carefully following the footsteps of Jesus.Thirdly, if the old nature cannot keep man away from that which is good, she will endeavor to spoil that which is good.
(1) At one time she will cause the thoughts to wander from one thing to the next.
(2) At another time there will be good thoughts which, however, will not be applicable at the moment. They are only fit to break the resolution toward that good thing which at that moment is to be performed.
(3) Again, ulterior motives and our ego can enter the picture which will hinder a person in his duty, causing him to lose his resolve and the stimulus to be removed; thus the purity of the duty is contaminated.
(4) Then there will be thoughts that all is devoid of the Spirit and but the work of nature yes, even hypocrisy.
(5) At another time the atheistic heart and unbelief come to the surface, which contaminate the performance of spiritual duty and instead of being refreshed by the performance of one’s duty, there is consternation and abhorrence that he has performed this good duty in such an evil manner. And thus the old nature agitates within.
Why I Love Riding in Arizona [Alpha and Omega Ministries] 2013-10-24 21:38
Just had to stop a couple of times today to snap a few pictures as I rode once again in South Mountain Park. After a pitch dark ascent long before sunrise, on my second run up Dobbins I saw this, and had to stop long enough to record it. Just glorious sunrises here in the Sonoran Desert.
And speaking of the Sonoran desert, here’s a later point in my ride, looking west. Arizona is truly a beautiful place. Blessed to live here.
Today on the Dividing Line: Continuationism, Cessationism. [Alpha and Omega Ministries] 2013-10-24 20:20
I decided to dedicate the entire program today to reviewing the first call Michael Brown took on The Line of Fire on Tuesday. It seemed to provide an excellent basis for actually addressing the most important elements of this controversy. I didn’t expect to take the whole hour, but, that’s what happened! Hopefully our listeners will find this a useful discussion. Here’s the audio of the program.
Here’s the video:
A Response to Dr. Shabir Ally (Part 2) [Alpha and Omega Ministries] 2013-10-24 20:17
Third, no writings survive from the disciples themselves. The Second Letter of Peter is admitted even by conservative scholars to be written after Peter’s death. The First Letter of Peter is disputed as to whether or not Peter wrote it. Some scholars think he wrote it; others think he did not. Hence we cannot rely on that letter either.
1) Dr. Ally is simply repeating standard skeptical lines without providing the necessary foundational evidence. This is how history is presented in academia today, but when you start asking tough questions, like, “Why?” you are often labelled a “fundamentalist” and ignored. If you ask why Petrine authorship is rejected, you get two lines of argumentation: A) the language of First and Second Peter differs greatly (a demonstrable fact known to anyone who has translated both), and 2) 2 Peter and Jude are clearly related and “late.” Why late? Well, you start with a particular theory of church development and then fit the early writing into your theory, of course. So, just as in arguing that the Pastoral Epistles are non-Pauline (because, we are told, the church situation seen therein did not “develop” until later–and how do we know that? We don’t. It’s a theory, though much of modern scholarship is loathe to admit such things) here we have an argument based upon an opinion regarding what topics could, and could not, have been being discussed in the primitive period of the church. These same mechanisms could be easily applied to the Qur’anic material as well. For example, all one has to do is theorize as to the nature of the development of the Islamic law over time and then, on that basis, assign certain surahs to a later period, after the life time of Muhammad, because they don’t “fit” with your theory! Of course, you could only get away with that in Western countries, currently.
N.B. I would like to provide some reading for those who wish to look more fully into these issues without overwhelming the reader with obscure references. Just a few resources that I grabbed quickly that would help to elucidate some of the preceding discussion:
For a full discussion of the relationship of Acts and the historical setting it presents (which would be directly relevant to Dr. Ally’s dismissal of the entirety of the recorded sermons as fabrications), see Colin J. Hember, The Book of Acts in the Setting of Hellenistic History (Eisenbraun’s, 1990). On the authorship of James, see Douglas Moo, The Letter of James in the Pillar New Testament Commentary Series, (Eerdmans, 2000), pp. 9-22. For a discussion of Pauline authorship of the Pastorals, see Hendriksen and Kistemaker, Thessalonians, the Pastorals, and Hebrews (Baker, 1955-1984), pp. 4-33. And for Petrine authorship issues relating to his epistolary literature, see Kistemaker, New Testament Commentary, Exposition of the Epistles of Peter and of the Epistle of Jude, (Baker, 1987), pp. 213-219.
Returning to Dr. Ally’s comments,
The Gospel of Matthew is now thought not to be from the disciple Matthew, since it is widely believed to be copied from Mark. The disciple Matthew is unlikely to have relied on the writing of a non-disciple, Mark, for information about Jesus.
This is surely the natural result of a slavish acceptance of a documentary dependence theory. (For a discussion of those issues, see Thomas and Farnell, The Jesus Crisis, Kregel, 1998). But once again we see the incoherence of a Muslim who believes in the inspiration of the Qur’an following such theoretical reconstructions of a document that, I would strongly argue, is the only one that can qualify to fulfill the referent in the Qur’an to the “Injeel.” What do I mean? Well, simply, if Dr. Ally is going to insist upon ignoring other possibilities as to the origination of the gospels, and insist that when Matthew and Mark both quote the same material, word for word, that this must show literary dependence (i.e., there can be no supernatural guidance to any human writing whatsoever), how does he then explain his own text, the Qur’an? As noted earlier, Dr. Ally believes the Qur’an accurately records the words of Jesus in a number of places when, in fact, historically, there is no connection between the Qur’an and the Jesus of history. The only possible explanation is supernatural inspiration, the very thing he rejects, a priori, for Matthew and Mark. So which will it be? And if similarity of language demands we have direct lineal literary relationship, does not the Qur’an likewise become guilty of the utilization of preceding sources in its references to Jewish myths and legends as well as gnostic gospels and other non-canonical Christian writings? Will Dr. Ally affirm that the author(s) of the Qur’an did, in fact, utilize pre-existing written documents? If so, which ones, we wonder?
As for the Gospel of John, this too cannot in its present form be credited to the disciple John. This Gospel went through stages of editing which I described in summary form as follows. The disciple John, Son of Zebedee preached his memories of Jesus. A disciple of John took John’s preaching and preached on it further. This disciple of the disciple eventually wrote the results of his preaching in the Gospel. As is generally known, preachers in the heat of their sermons tend to mix up the quoted material with their own explanations. This is what happened also when this disciple of the disciple preached. This explains why in John’s Gospel it is often difficult to know where the quoted words of Jesus end and where the commentary of the writer begins. Moreover, a later editor inserted parts into the Gospel, and added the last chapter as well. In sum we have no dependable first-hand writing of the original disciples of Jesus.
I would simply like to ask Dr. Ally to provide us with some documentation of this amazingly complex reconstruction. This is form criticism taken to the level of fantasy, as is so often seen in the most destructive of liberal critics. But where is the evidence of this fanciful history? And why can’t it be applied with equal facility to Surah al-Baqara, for example? If there were a series of redactions, why do we not have multiple transmission streams for this gospel? But what we really must see is how easily Shabir goes from a series of unfounded speculations to an absolutely firm conclusion, “we have no dependable first-hand writings of the original disciples of Jesus.” Hopefully the reader can see that the chasm of evidence Dr. Ally has leapt with that statement is far wider than can be traversed successfully.
One other question I would honestly ask of the unbiased reader of the Qur’an: do you really think the author of the Qur’an would have come to the same conclusion Dr. Ally has?
The fourth point Dr. Ally raised is very important and I do not wish to rush through it, as it provides an excellent example of how destructive skeptical criticism can force a text into a form never dreamed of by earlier generations, let alone by the author himself. As this month is the heaviest travel month of my adult life (thus far!), and I leave for Canada (ironically) in the morning, I will need to continue my response as time allows. Thank you for reading!
Speaking of The Extraordinary Spiritual Gifts [Alpha and Omega Ministries] 2013-10-24 10:46
Some of you may be interested in a lecture that Dr. George Knight gave a few years ago titled “Cessation of the Extraordinary Spiritual Gifts.” He begins by stating “I am not in favor of the cessation of the spiritual gifts. None of us should be….” Click here to listen.
The lecture was published in The Beauty and Glory of the Holy Spirit
BTW: George Knight, III is the author of perhaps the best commentary on the pastoral epistles. Click here for more information. Grab the hardcover while you can, because it is now being printed in paperback.
The Bible, the Spirit’s Masterpiece [Shepherd Press] 2013-10-30 15:28
Good writing is a work of connected beauty. Words and thoughts are woven together that build word pictures that can be etched into hearts. No other piece of literature is comparable to the Bible in this regard. When we read we must look for these connections to understand the power of the Spirit’s work. The Bible is the literary masterpiece of all time. Every word is exactly placed to draw the hearts of God’s people to the life that is truly life.
It can be dangerous to make connections that are not obvious in the text. But it is equally dangerous to ignore the connections that are there. Like an exquisitely cut diamond, the Bible is a multi-faceted literary gem of the highest quality. The purest treasures are found by longing to have God’s Spirit open them to us as his word is read, mediated upon and embraced.
Here is just one example of the thousands of such connections. In James 1:2-3 we read:
Count it all joy, my brothers, when you meet trials of various kinds, for you know that the testing of your faith produces steadfastness.
These words are rightly used to encourage when difficulties come. But remember that these words are part of a letter with a clear mission to bring hope to God’s people in distress. Don’t just select a fragment and think these few words are all that God has for trials. Look for the connection in the text to enhance and strengthen these words. Look eagerly for how these words in the opening of James connect to other portions in the book and to the whole of Scripture. Meditating on Scripture is not like the eastern, mystic practice of saying a mantra over and over again. Biblical mediation is a search for the rich provision of God’s Spirit in his word.
So we should not be surprised when just a few sentences later we again read about trials and steadfastness in verse 12:
Blessed is the man who remains steadfast under trial, for when he has stood the test he will receive the crown of life, which God has promised to those who love him.
We are to count trials as joy because they lead us to the crown of life which God has promised. Steadfastness is not being stoic. Rather being steadfast anticipates the wonder of God’s promises. This is a true reason for joy.
Appreciate the Bible for the intricate, precious masterpiece that it is. It was crafted by the Holy Spirit for glory of God and our good. It can and will bring healing to the deepest parts of your soul.
Halloween – the ultimate trick or treat [Shepherd Press] 2013-10-29 16:42
Halloween is viewed by many as a night of family fun, fantasy dress up, and sweet rewards. Millions participate in Halloween festivities for these reasons. If this was all there was to Halloween there would be nothing to be concerned about. But not everything is at it appears to be. There is a dark-side to Halloween.
The last few weeks cable channels have flooded TV screens with horror movies specializing in blood, fear, and gore. Modern imaging technology makes these films shockingly realistic. Satanists claim Halloween as their holy day. The occult revels in this dark celebration.
The contrast is stunning. One side of the Halloween coin is bright, fun and exciting. The other side represents the darkest of features of fallen man. Both sides are celebrated on the same night. Is it possible to ignore the dark side of Halloween and enjoy only the fun part?
I believe one of the real dangers of Halloween is to classify the darkness associated with the day as fantasy that can be ignored. But make no mistake, the forces of darkness are real and predatory. Numerous passages of the Bible warn of spiritual warfare. The enemy is real and vicious. The Bible warns us not to be taken in and to avoid evil.
Does this mean that you should not help your children dress up as pirates or princesses and visit your neighbors looking for goodies? Not necessarily. But have you counted the cost? Remember, the theme of your spiritual enemy is deception. His goal is the destruction of you and your family. Some claim that Halloween is his night.
For example, consider the phrase, “trick or treat”. Sounds harmless enough. It is just an expression to gain some candy. But what is Biblical but the phrase? What it really means is that if I don’t get a treat, you can expect a trick against you. Now I know most of you reading this are not teaching your children to mess up the neighbor’s lawn or house if they are not sufficiently rewarded. But then, why even utter the phrase? Is this honoring God? It is easy to say it is just an expression, but is it an expression that brings honor to God?
Think carefully about Halloween. Remember it is a two-sided coin The dark side is not pretty. You can hope that by just looking at the fun side that the darkness can be ignored. Is this really what you want to teach your children; just ignore the bad things and focus on the good? After all, Halloween is so much fun. How many other things are there that present a positive upside but when you turn the coin over you are greeted by the smiling face of the enemy? I am not attempting to insist on a particular course of action. I am just praying that you will remember that since the fall things are often not what they appear to be.
Stories for Life [Shepherd Press] 2013-10-29 02:22
Man was made for stories. We remember stories. We laugh at stories. We cry over stories. We are motivated by stories. Why? Because God made us to love stories. The legacy of a culture is told by narratives, not by encyclopedias. Even in a culture as bent on moving away from God as our current one is, there are still biblical stories etched into the minds of people in our culture. These biblical narratives transcend ideological barriers. The Prodigal Son, The Good Samaritan, the Christmas Story, and others speak of a God who may not be as easily dismissed as cultural elitists believe. While these narratives are often marred and confused in the culture, they still remain, and they still bring conviction to people. Themes of lostness, hope, and redemption resonate with the human spirit. For those who do not know Christ, these great themes may serve only to disturb rather than comfort–but still, people are drawn to stories. Examine the lists of the all-time most popular movies. The one thing these movies all have in common is that they tell memorable stories.
Because the biblical narratives are so memorable it is important that we interpret them correctly. Biblical narratives are designed by the Holy Spirit to communicate a particular theme or truth. This does not mean that every point in the narrative provides an example to be followed or implemented. Sometimes the narratives communicate actions that should most definitely not be copied. For example, the story of Simeon and Levi taking revenge for the way their sister Dinah was treated by the Shechemites is not a model for us to follow. It is a true story, but it is not meant to be an example of godly behavior. The brothers should have sought their father’s direction before taking action. These narratives must be read with discernment. Even when the narratives do contain examples of godly behavior, not everything in the story should be followed. Gideon showed courage in following God by attacking the vastly superior numbers of the Midianite army with only 300 men. However, he is often remembered more for laying out of a fleece to see if God’s word was really true. The story of Gideon is not meant to be an example of how to get divine guidance. Rather, the story demonstrates that God is faithful even when we are not.
Narratives are powerful, but they must be read carefully, and it is important not to draw more from narratives than the Holy Spirit intends. But it is equally important to intimately know the stories of the Bible. You and your children need to know them. These narratives are yours for life.
Off for a week [Shepherd Press] 2013-10-22 00:57
I am going to take the next week off to spend time with my family after the death of my wife. Thank you for the encouragement that so many of you have expressed. May the grace of God continue to bless and keep all of us in his good care.
Jay Younts
Marketing Strategy [Shepherd Press] 2013-10-21 10:56
Let’s evaluate the strategies of enlisting recruits of the two opposing forces involved in spiritual warfare. We will start with the plan of the Kingdom of Heaven. It is a straightforward approach and could be stated like this:
Life is to be lived for the glory and honor of God.
Man has rebelled against that reality and is in need of rescue.
Strictly on his own initiative, through the sacrificial death of his son, God rescues people from darkness and brings them into the kingdom of light for all eternity.
That is the strategy of one side. Here is the strategy of the other side, the kingdom of this world. It is also straightforward.
Satan hates mankind and has a wretched plan that ends not in rescue but in death and everlasting torment.
People must be deceived so that they will not recognize the purpose of this plan until it is too late.
If both sides adhered to the practice of full-disclosure, the kingdom of darkness would be at a clear disadvantage.
Satan, of course, realized that he dare not tell the truth about his intentions. So, he told Eve that following his advice would make her wise, delight her senses, and make her on par with her creator. Eve and her husband bought the lie. Satan knew that by enlisting our first parents into his kingdom all of mankind that followed would be serving him and not God.
However, Satan is not God. Satan does not have the power of Christ. The power of the gospel is greater than his lies. Though Satan continues to deceive, he is only able to offer things that have to do with this world. All of his promises are connected to this life; his rewards are from a world that is passing away. He knows he must make his promises attractive for this life because, beyond this life, he can offer nothing.
Thus, Satan’s marketing strategy has to do with finding accomplishment and satisfaction in this life. He is shamelessly deceptive: Wealth offers security. An unwanted pregnancy will ruin your life as well as the baby. The acceptance and praise of others will meet our deepest relational cravings. Education will make one truly wise. Sex is meant to be enjoyed by all and is our biological right to indulge our flesh in any way that is pleasing. Athletic and business achievements will provide a lifetime of recognition and satisfaction. Self-pleasure is the ultimate pursuit. The beauty and complexity of nature is worth a lifetime of devotion. It is impossible to have too much of a good thing.
All of these deceptions are designed to do one thing and one thing only: hide the reality of death and the torture of hell. You see, Satan must distract his subjects from the horror that lies just beyond the doorway of death. (Ephesians 2:1-3)
Even Christians can be deceived by the promises of the enemy. Even though Satan knows he cannot win us back to his side, he knows that our wrong desires can help him in his grand plan of deception. Ask God for the clarity of vision to help expose the deception of the promises of this world. If you live for these deceptions, you will not help your children or those around you to see the ugliness of a strategy designed to lead straight to hell.
Life makes you weary, Jesus brings rest [Shepherd Press] 2013-10-21 04:01
The God who sustains the universe by his mighty power also cares for you. Do not disconnect your weary moments from the rest of your life. God is there for all of it.
The truth that you will never hear on the 6 p.m. news is that since the Fall, life itself will make you weary; Every other reason under the sun will be given to explain why you are weary. But the one reason that really matters is lost in political correctness—your weariness is part of the impact of sin and the resulting curse.
The truth is that successes are muted by failures. Happiness is often quickly followed by sorrow. Good days and bad days intermingle. Sin reaches deep into our lives and into our souls. In all of this Jesus says to come to him and he will give you rest. He is not speaking only of heaven, the ultimate rest. Christ is speaking of knowing him in the weariness of everyday life. Right now!
Jesus makes this audacious—and yet wonderful promise—I will give you rest! Believe what he says! Pray you will see the sufficiency of your savior’s love through his word in all that you do.
Trust the word of Christ:
28 “Come to me, all you who are weary and burdened, and I will give you rest. 29 Take my yoke upon you and learn from me, for I am gentle and humble in heart, and you will find rest for your souls. 30 For my yoke is easy and my burden is light.”
Matthew 11:28-30
Thought for the Lord’s Day [Shepherd Press] 2013-10-19 23:52
Because of the commitment of God to his word and his covenant, we have a sure hope.
My wife, Ruth, was a faithful servant of her Lord. The Lord did redeem her as he promised. Today that promise was kept as Ruth went to be with her God as she left behind her dead body and entered into the wonder of eternity. The hope of heaven is not a random one. It is made sure because of the commitment of God. May this hope motivate you and change you in all that you are and do.
“Remember these things, O Jacob,
and Israel, for you are my servant;
I formed you; you are my servant;
O Israel, you will not be forgotten by me.
I have blotted out your transgressions like a cloud
and your sins like mist;
return to me, for I have redeemed you.
Sing, O heavens, for the Lord has done it;
shout, O depths of the earth;
break forth into singing, O mountains,
O forest, and every tree in it!
For the Lord has redeemed Jacob,
and will be glorified in Israel.”
From Isaiah 44
The Lion of Judah [Shepherd Press] 2013-10-18 22:25
If we are to live a life that is wise, it is imperative that we understand we have an Enemy who attempts to discredit the wonders of God with counterfeit musings. This discrediting can take the form of misinterpreting the good things that God does. For example, as magnificent as a sunset may be, it is not the ultimate that God does. That ultimate is reserved for heaven and the new earth. But the enemy wants you to think that what you see now is all there is.
Also, we have grown accustomed to seeing breathtaking imagery on the big screen. But even these images pale in comparison to the reality of the world that remains unseen to us. Plus, even when we see these images, we know they are the results of skilled electronic artists. But think with me for a moment about imagery that is not computer generated, imagery that is more real than anything we can imagine. For example, here is a description of the ultimate reality of an event in God’s universe, with drama that exceeds our own imagination:
“Then I saw in the right hand of him who sat on the throne a scroll with writing on both sides and sealed with seven seals. And I saw a mighty angel proclaiming in a loud voice, “Who is worthy to break the seals and open the scroll?” But no one in heaven or on earth or under the earth could open the scroll or even look inside it. I wept and wept because no one was found who was worthy to open the scroll or look inside. Then one of the elders said to me, “Do not weep! See, the Lion of the tribe of Judah, the Root of David, has triumphed. He is able to open the scroll and its seven seals.””
Revelation 5:1-5
My daughter read this passage to my wife this evening. My wife’s body is giving way to the war of this world. From a human perspective she has only days and perhaps just hours left on this earth. Yet a greater, more magnificent drama of awe awaits her. Creatures and her Savior undaunted or spoiled by sin are moments away. Her enemy wants us to discredit the wonder of what God has for his people. But this passage and others like it have only served to reassure Ruth that greater things are in store for those purchased by the Lamb.
Don’t buy the lie that the wonder of this planet is all that there is. The mighty One who sits on the throne will offer wonders and beauty beyond what we can know here. This pure vision drives my wife’s hunger for heaven. I only pray that it will drive mine as well.
Understand your limitations [Shepherd Press] 2013-10-18 01:59
Clint Eastwood may not be your first choice for understanding truth. But there is one quote of his that does resonate with biblical truth. Eastwood says “a man has got to understand his limitations.” Proverbs 3:7 says “don’t be wise in your own eyes.” In other words if you think your own understanding is something you can rely on, you are headed for trouble.
The structure of Hebrew wisdom literature, of which Proverbs is a part, uses a literary device called parallelism to highlight important truths. In this case, being wise in your own eyes is contrasted exercising the fear of the Lord and turning from evil. If you think you can trust yourself, you will not exhibit a healthy fear of God and thus, you will not shun evil. Being wise in your own eyes will lead to a lack of health and nourishment for your body.
There are more than a few ways that this impacts Christians. There are subtle, but impactful examples of the damage done by not understanding our limitations. When I try to do what only God can do, I am being wise in my own eyes. When I worry about how to control things that are in God’s control, I become wise in my own eyes. I fail to understand my limitations and do not fear God. When this happens my body reacts to the stress of taking on things which only God controls. Thus, the New Testament commands in the strongest terms that we not worry.
One way to address stress is to embrace the fear of the great God of Heaven who has promised to care for us. Psalm 23:1 teaches that since the Lord is our Shepherd, we have no needs. When we live as though our Shepherd is doing a bad job of caring for us, stress inevitably follows. Our children, who are profoundly influenced by our example, come to know the dangers of stress and the failure of understanding our God-given limitations. Then, they too will be come wise in their own eyes. In humility, teach your children to know their own limitations. Once this happens, they, like us, can know the wonder of the God who has no limitations!
Safe Sex, an urban legend [Shepherd Press] 2013-10-17 02:30
Teaching your children to honor God’s authority by obeying quickly and pleasantly has many blessings. One of these blessings is to learn to avoid the world’s wisdom, no matter how attractive it may appear. For example, there is the lie that there can be safe sex outside of marriage.
Modern man, in his proverbial darkened state, plunges ever deeper into darkness by seeking new and creative ways to say YES to sex outside of marriage. This thinking brings a rising tide of the health risks and emotional wreckage that comes from the practice of “safe sex.”
Safe sex outside of marriage is a deadly oxymoron of modern life. The truth is that any sex outside of marriage is anything but safe! Extramarital sex is one of the most dangerous activities that people can engage in. A few moments of pleasure are stupidly traded for a life of worry, insecurity and physical danger. Within one generation, the slide into immorality has gone from can I get her to kiss me on the first date? to can I … well, you know what follows. The words of Solomon, 3,000 years ago, still ring true:
Can a man scoop fire into his lap
without his clothes being burned?
Can a man walk on hot coals
without his feet being scorched?
So is he who sleeps with another man’s wife;
no one who touches her will go unpunished. Proverbs 6:27-29
As Romans says, the truth of God has been exchanged for a lie. In this case, the lie is that there is safe sex outside of marriage. For example, a young couple, using certain types of contraceptives, may, in fact, prevent the spread of a certain type of disease or infection or a pregnancy. But what about the spiritual damage that is being done every time this young couple engages in “protected” sexual activity. Their very souls are laid open to be ravaged by lies, deceit and insecurity. They are being wounded with wounds that may never heal. They will learn the fleeting enticements of self-pleasure instead of the treasures of sensitivity and self-less love. Relationships that have “protected” sex are plagued with fear, doubt, and even multiple forms of abuse. Divorce rates are up, spousal and domestic abuse is rampant, and abortions are considered to be a necessary part of life. This is the grim harvest of safe sex.
The world teaches that you can have sex anyway, anytime you want to. Safe, protected sex outside of marriage is a modern urban legend, a hideous lie that waits to devour your children. Christian, have the courage to tell your children the truth about what constitutes sex that is safe. The marriage bed alone is the one place where sex is truly protected and is truly safe.
Determination of genomic copy number alteration emphasizing a restriction site-based strategy of genome re-sequencing [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Copy number abbreviation (CNA) is one type of genomic aberration that is often induced by genome instability and is associated with diseases such as cancer. Determination of the genome-wide CNA profile is an important step in identifying the underlying mutation mechanisms. Genomic data based on next-generation sequencing technology are particularly suitable for determination of high-quality CNA profile. Now is an important time to reevaluate the use of sequencing techniques for CNA analysis, especially with the rapid growth of the different targeted genome and whole-genome sequencing strategies.
Results: In this study, we provide a comparison of resequencing strategies, with regard to their utility, applied to the same hepatocellular carcinoma sample for copy number determination. These strategies include whole-genome, exome and restriction site-associated DNA (RAD) sequencing. The last of these strategies is a targeted sequencing technique that involves cutting the genome with a restriction enzyme and isolating the targeted sequences. Our data demonstrate that RAD sequencing is an efficient and comprehensive strategy that allows the cost-effective determination of CNAs. Further investigation of RAD sequencing data led to the finding that a precise measurement of the allele frequency would be a helpful complement to the read depth for CNA analysis for two reasons. First, knowledge of the allele frequency helps to resolve refined calculations of allele-specific copy numbers, which, in turn, identify the functionally important CNAs that are under natural selection on the parental alleles. Second, this knowledge enables deconvolution of CNA patterns in complex genomic regions.
Contact: juncai@big.ac.cn or ciwu@uchicago.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
The code structure of the p53 DNA-binding domain and the prognosis of breast cancer patients [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: The tumor-suppressor gene TP53 mutations are diverse in the central region encoding the DNA-binding domain. It has not been clear whether the prognostic significance for survival in breast cancer patients is the same for all types of mutations. Are there specific types of mutations carrying a worse prognosis? To understand the correlation between the mutations in the gene encoding the DNA-binding domain and the prognosis of breast cancer, we studied the code structure of the DNA-binding domain of breast cancer patients by using various artificial codes in information transmission.
Results: We indicated that the prognostic significance of all types of mutations in the DNA-binding domain is not the same, and that the DNA-binding domain having a certain code structure is important for estimating the prognosis of breast cancer patients.
Exploring variation-aware contig graphs for (comparative) metagenomics using MaryGold [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Although many tools are available to study variation and its impact in single genomes, there is a lack of algorithms for finding such variation in metagenomes. This hampers the interpretation of metagenomics sequencing datasets, which are increasingly acquired in research on the (human) microbiome, in environmental studies and in the study of processes in the production of foods and beverages. Existing algorithms often depend on the use of reference genomes, which pose a problem when a metagenome of a priori unknown strain composition is studied. In this article, we develop a method to perform reference-free detection and visual exploration of genomic variation, both within a single metagenome and between metagenomes.
Results: We present the MaryGold algorithm and its implementation, which efficiently detects bubble structures in contig graphs using graph decomposition. These bubbles represent variable genomic regions in closely related strains in metagenomic samples. The variation found is presented in a condensed Circos-based visualization, which allows for easy exploration and interpretation of the found variation.
We validated the algorithm on two simulated datasets containing three respectively seven Escherichia coli genomes and showed that finding allelic variation in these genomes improves assemblies. Additionally, we applied MaryGold to publicly available real metagenomic datasets, enabling us to find within-sample genomic variation in the metagenomes of a kimchi fermentation process, the microbiome of a premature infant and in microbial communities living on acid mine drainage. Moreover, we used MaryGold for between-sample variation detection and exploration by comparing sequencing data sampled at different time points for both of these datasets.
Availability: MaryGold has been written in C++ and Python and can be downloaded from http://bioinformatics.tudelft.nl/software
Contact: d.deridder@tudelft.nl
A statistical variant calling approach from pedigree information and local haplotyping with phase informative reads [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Variant calling from genome-wide sequencing data is essential for the analysis of disease-causing mutations and elucidation of disease mechanisms. However, variant calling in low coverage regions is difficult due to sequence read errors and mapping errors. Hence, variant calling approaches that are robust to low coverage data are demanded.
Results: We propose a new variant calling approach that considers pedigree information and haplotyping based on sequence reads spanning two or more heterozygous positions termed phase informative reads. In our approach, genotyping and haplotyping by the assignment of each read to a haplotype based on phase informative reads are simultaneously performed. Therefore, positions with low evidence for heterozygosity are rescued by phase informative reads, and such rescued positions contribute to haplotyping in a synergistic way. In addition, pedigree information supports more accurate haplotyping as well as genotyping, especially in low coverage regions. Although heterozygous positions are useful for haplotyping, homozygous positions are not informative and weaken the information from heterozygous positions, as majority of positions are homozygous. Thus, we introduce latent variables that determine zygosity at each position to filter out homozygous positions for haplotyping. In performance evaluation with a parent–offspring trio sequencing data, our approach outperforms existing approaches in accuracy on the agreement with single nucleotide polymorphism array genotyping results. Also, performance analysis considering distance between variants showed that the use of phase informative reads is effective for accurate variant calling, and further performance improvement is expected with longer sequencing data.
Contact: nagasaki@megabank.tohoku.ac.jp or kojima@megabank.tohoku.ac.jp
Supplementary information: Supplementary data are available at Bioinformatics online.
Assessing the validity and reproducibility of genome-scale predictions [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Validation and reproducibility of results is a central and pressing issue in genomics. Several recent embarrassing incidents involving the irreproducibility of high-profile studies have illustrated the importance of this issue and the need for rigorous methods for the assessment of reproducibility.
Results: Here, we describe an existing statistical model that is very well suited to this problem. We explain its utility for assessing the reproducibility of validation experiments, and apply it to a genome-scale study of adenosine deaminase acting on RNA (ADAR)-mediated RNA editing in Drosophila. We also introduce a statistical method for planning validation experiments that will obtain the tightest reproducibility confidence limits, which, for a fixed total number of experiments, returns the optimal number of replicates for the study.
Availability: Downloadable software and a web service for both the analysis of data from a reproducibility study and for the optimal design of these studies is provided at http://ccmbweb.ccv.brown.edu/reproducibility.html
Contact: Charles_Lawrence@Brown.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
INSECT: IN-silico SEarch for Co-occurring Transcription factors [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Transcriptional regulation occurs through the concerted actions of multiple transcription factors (TFs) that bind cooperatively to cis-regulatory modules (CRMs) of genes. These CRMs usually contain a variable number of transcription factor-binding sites (TFBSs) involved in related cellular and physiological processes. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been effective in detecting TFBSs and nucleosome location to identify potential CRMs in genome-wide studies. Although several attempts were previously reported to predict the potential binding of TFs at TFBSs within CRMs by comparing different ChIP-seq data, these have been hampered by excessive background, usually emerging as a consequence of experimental conditions. To understand these complex regulatory circuits, it would be helpful to have reliable and updated user-friendly tools to assist in the identification of TFBSs and CRMs for gene(s) of interest.
Results: Here we present INSECT (IN-silico SEarch for Co-occurring Transcription factors), a novel web server for identifying potential TFBSs and CRMs in gene sequences. By combining several strategies, INSECT provides flexible analysis of multiple co-occurring TFBSs, by applying differing search schemes and restriction parameters.
Availability and implementation: INSECT is freely available as a web server at http://bioinformatics.ibioba-mpsp-conicet.gov.ar/INSECT
Contact: cperezcastro@ibioba-mpsp-conicet.gov.ar or pyankilevich@ibioba-mpsp-conicet.gov.ar
Supplementary information: Supplementary data are available at Bioinformatics online.
PyroHMMvar: a sensitive and accurate method to call short indels and SNPs for Ion Torrent and 454 data [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: The identification of short insertions and deletions (indels) and single nucleotide polymorphisms (SNPs) from Ion Torrent and 454 reads is a challenging problem, essentially because these techniques are prone to sequence erroneously at homopolymers and can, therefore, raise indels in reads. Most of the existing mapping programs do not model homopolymer errors when aligning reads against the reference. The resulting alignments will then contain various kinds of mismatches and indels that confound the accurate determination of variant loci and alleles.
Results: To address these challenges, we realign reads against the reference using our previously proposed hidden Markov model that models homopolymer errors and then merges these pairwise alignments into a weighted alignment graph. Based on our weighted alignment graph and hidden Markov model, we develop a method called PyroHMMvar, which can simultaneously detect short indels and SNPs, as demonstrated in human resequencing data. Specifically, by applying our methods to simulated diploid datasets, we demonstrate that PyroHMMvar produces more accurate results than state-of-the-art methods, such as Samtools and GATK, and is less sensitive to mapping parameter settings than the other methods. We also apply PyroHMMvar to analyze one human whole genome resequencing dataset, and the results confirm that PyroHMMvar predicts SNPs and indels accurately.
Availability and implementation: Source code freely available at the following URL: https://code.google.com/p/pyrohmmvar/, implemented in C++ and supported on Linux.
Contact: ruijiang@tsinghua.edu.cn or cengf08@mails.thu.edu.cn
A general species delimitation method with applications to phylogenetic placements [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Sequence-based methods to delimit species are central to DNA taxonomy, microbial community surveys and DNA metabarcoding studies. Current approaches either rely on simple sequence similarity thresholds (OTU-picking) or on complex and compute-intensive evolutionary models. The OTU-picking methods scale well on large datasets, but the results are highly sensitive to the similarity threshold. Coalescent-based species delimitation approaches often rely on Bayesian statistics and Markov Chain Monte Carlo sampling, and can therefore only be applied to small datasets.
Results: We introduce the Poisson tree processes (PTP) model to infer putative species boundaries on a given phylogenetic input tree. We also integrate PTP with our evolutionary placement algorithm (EPA-PTP) to count the number of species in phylogenetic placements. We compare our approaches with popular OTU-picking methods and the General Mixed Yule Coalescent (GMYC) model. For de novo species delimitation, the stand-alone PTP model generally outperforms GYMC as well as OTU-picking methods when evolutionary distances between species are small. PTP neither requires an ultrametric input tree nor a sequence similarity threshold as input. In the open reference species delimitation approach, EPA-PTP yields more accurate results than de novo species delimitation methods. Finally, EPA-PTP scales on large datasets because it relies on the parallel implementations of the EPA and RAxML, thereby allowing to delimit species in high-throughput sequencing data.
Availability and implementation: The code is freely available at www.exelixis-lab.org/software.html.
Contact: Alexandros.Stamatakis@h-its.org
Supplementary information: Supplementary data are available at Bioinformatics online.
A new statistic for identifying batch effects in high-throughput genomic data that uses guided principal component analysis [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Batch effects are due to probe-specific systematic variation between groups of samples (batches) resulting from experimental features that are not of biological interest. Principal component analysis (PCA) is commonly used as a visual tool to determine whether batch effects exist after applying a global normalization method. However, PCA yields linear combinations of the variables that contribute maximum variance and thus will not necessarily detect batch effects if they are not the largest source of variability in the data.
Results: We present an extension of PCA to quantify the existence of batch effects, called guided PCA (gPCA). We describe a test statistic that uses gPCA to test whether a batch effect exists. We apply our proposed test statistic derived using gPCA to simulated data and to two copy number variation case studies: the first study consisted of 614 samples from a breast cancer family study using Illumina Human 660 bead-chip arrays, whereas the second case study consisted of 703 samples from a family blood pressure study that used Affymetrix SNP Array 6.0. We demonstrate that our statistic has good statistical properties and is able to identify significant batch effects in two copy number variation case studies.
Conclusion: We developed a new statistic that uses gPCA to identify whether batch effects exist in high-throughput genomic data. Although our examples pertain to copy number data, gPCA is general and can be used on other data types as well.
Availability and implementation: The gPCA R package (Available via CRAN) provides functionality and data to perform the methods in this article.
Contact: reesese@vcu.edu or eckel@mayo.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
A-clustering: a novel method for the detection of co-regulated methylation regions, and regions associated with exposure [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: DNA methylation is a heritable modifiable chemical process that affects gene transcription and is associated with other molecular markers (e.g. gene expression) and biomarkers (e.g. cancer or other diseases). Current technology measures methylation in hundred of thousands, or millions of CpG sites throughout the genome. It is evident that neighboring CpG sites are often highly correlated with each other, and current literature suggests that clusters of adjacent CpG sites are co-regulated.
Results: We develop the Adjacent Site Clustering (A-clustering) algorithm to detect sets of neighboring CpG sites that are correlated with each other. To detect methylation regions associated with exposure, we propose an analysis pipeline for high-dimensional methylation data in which CpG sites within regions identified by A-clustering are modeled as multivariate responses to environmental exposure using a generalized estimating equation approach that assumes exposure equally affects all sites in the cluster. We develop a correlation preserving simulation scheme, and study the proposed methodology via simulations. We study the clusters detected by the algorithm on high dimensional dataset of peripheral blood methylation of pesticide applicators.
Availability: We provide the R package Aclust that efficiently implements the A-clustering and the analysis pipeline, and produces analysis reports. The package is found on http://www.hsph.harvard.edu/tamar-sofer/packages/
Contact: tsofer@hsph.harvard.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Strengths and limitations of microarray-based phenotype prediction: lessons learned from the IMPROVER Diagnostic Signature Challenge [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: After more than a decade since microarrays were used to predict phenotype of biological samples, real-life applications for disease screening and identification of patients who would best benefit from treatment are still emerging. The interest of the scientific community in identifying best approaches to develop such prediction models was reaffirmed in a competition style international collaboration called IMPROVER Diagnostic Signature Challenge whose results we describe herein.
Results: Fifty-four teams used public data to develop prediction models in four disease areas including multiple sclerosis, lung cancer, psoriasis and chronic obstructive pulmonary disease, and made predictions on blinded new data that we generated. Teams were scored using three metrics that captured various aspects of the quality of predictions, and best performers were awarded. This article presents the challenge results and introduces to the community the approaches of the best overall three performers, as well as an R package that implements the approach of the best overall team.
The analyses of model performance data submitted in the challenge as well as additional simulations that we have performed revealed that (i) the quality of predictions depends more on the disease endpoint than on the particular approaches used in the challenge; (ii) the most important modeling factor (e.g. data preprocessing, feature selection and classifier type) is problem dependent; and (iii) for optimal results datasets and methods have to be carefully matched. Biomedical factors such as the disease severity and confidence in diagnostic were found to be associated with the misclassification rates across the different teams.
Availability: The lung cancer dataset is available from Gene Expression Omnibus (accession, GSE43580). The maPredictDSC R package implementing the approach of the best overall team is available at www.bioconductor.org or http://bioinformaticsprb.med.wayne.edu/.
Contact: gustavo@us.ibm.com
Supplementary information: Supplementary data are available at Bioinformatics online.
GIM3E: condition-specific models of cellular metabolism developed from metabolomics and expression data [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Genome-scale metabolic models have been used extensively to investigate alterations in cellular metabolism. The accuracy of these models to represent cellular metabolism in specific conditions has been improved by constraining the model with omics data sources. However, few practical methods for integrating metabolomics data with other omics data sources into genome-scale models of metabolism have been developed.
Results: GIM3E (Gene Inactivation Moderated by Metabolism, Metabolomics and Expression) is an algorithm that enables the development of condition-specific models based on an objective function, transcriptomics and cellular metabolomics data. GIM3E establishes metabolite use requirements with metabolomics data, uses model-paired transcriptomics data to find experimentally supported solutions and provides calculations of the turnover (production/consumption) flux of metabolites. GIM3E was used to investigate the effects of integrating additional omics datasets to create increasingly constrained solution spaces of Salmonella Typhimurium metabolism during growth in both rich and virulence media. This integration proved to be informative and resulted in a requirement of additional active reactions (12 in each case) or metabolites (26 or 29, respectively). The addition of constraints from transcriptomics also impacted the allowed solution space, and the cellular metabolites with turnover fluxes that were necessarily altered by the change in conditions increased from 118 to 271 of 1397.
Availability: GIM3E has been implemented in Python and requires a COBRApy 0.2.x. The algorithm and sample data described here are freely available at: http://opencobra.sourceforge.net/
Contacts: brianjamesschmidt@gmail.com or hyduke@usu.edu
Supplementary information: Supplementary information is available at Bioinformatics online.
DNorm: disease name normalization with pairwise learning to rank [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Despite the central role of diseases in biomedical research, there have been much fewer attempts to automatically determine which diseases are mentioned in a text—the task of disease name normalization (DNorm)—compared with other normalization tasks in biomedical text mining research.
Methods: In this article we introduce the first machine learning approach for DNorm, using the NCBI disease corpus and the MEDIC vocabulary, which combines MeSH® and OMIM. Our method is a high-performing and mathematically principled framework for learning similarities between mentions and concept names directly from training data. The technique is based on pairwise learning to rank, which has not previously been applied to the normalization task but has proven successful in large optimization problems for information retrieval.
Results: We compare our method with several techniques based on lexical normalization and matching, MetaMap and Lucene. Our algorithm achieves 0.782 micro-averaged F-measure and 0.809 macro-averaged F-measure, an increase over the highest performing baseline method of 0.121 and 0.098, respectively.
Availability: The source code for DNorm is available at http://www.ncbi.nlm.nih.gov/CBBresearch/Lu/Demo/DNorm, along with a web-based demonstration and links to the NCBI disease corpus. Results on PubMed abstracts are available in PubTator: http://www.ncbi.nlm.nih.gov/CBBresearch/Lu/Demo/PubTator
Contact: zhiyong.lu@nih.gov
Quantifying the complexity of medical research [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: A crucial phenomenon of our times is the diminishing marginal returns of investments in pharmaceutical research and development. A potential reason is that research into diseases is becoming increasingly complex, and thus more burdensome, for humans to handle. We sought to investigate whether we could measure research complexity by analyzing the published literature.
Results: Through the text mining of the publication record of multiple diseases, we have found that the complexity and novelty of disease research has been increasing over the years. Surprisingly, we have also found that research on diseases with higher publication rate does not possess greater complexity or novelty than that on less-studied diseases. We have also shown that the research produced about a disease can be seen as a differentiated area of knowledge within the wider biomedical research. For our analysis, we have conceptualized disease research as a parallel multi-agent search in which each scientific agent (a scientist) follows a search path based on a model of a disease. We have looked at trends in facts published for diseases, measured their diversity and turnover using the entropy measure and found similar patterns across disease areas.
Contact: raul.rodriguez-esteban@roche.com
DIST: direct imputation of summary statistics for unmeasured SNPs [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Genotype imputation methods are used to enhance the resolution of genome-wide association studies, and thus increase the detection rate for genetic signals. Although most studies report all univariate summary statistics, many of them limit the access to subject-level genotypes. Because such an access is required by all genotype imputation methods, it is helpful to develop methods that impute summary statistics without going through the interim step of imputing genotypes. Even when subject-level genotypes are available, due to the substantial computational cost of the typical genotype imputation, there is a need for faster imputation methods.
Results: Direct Imputation of summary STatistics (DIST) imputes the summary statistics of untyped variants without first imputing their subject-level genotypes. This is achieved by (i) using the conditional expectation formula for multivariate normal variates and (ii) using the correlation structure from a relevant reference population. When compared with genotype imputation methods, DIST (i) requires only a fraction of their computational resources, (ii) has comparable imputation accuracy for independent subjects and (iii) is readily applicable to the imputation of association statistics coming from large pedigree data. Thus, the proposed application is useful for a fast imputation of summary results for (i) studies of unrelated subjects, which (a) do not provide subject-level genotypes or (b) have a large size and (ii) family association studies.
Availability and implementation: Pre-compiled executables built under commonly used operating systems are publicly available at http://code.google.com/p/dist/.
Contact: dlee4@vcu.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
catRAPID omics: a web server for large-scale prediction of protein-RNA interactions [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: Here we introduce catRAPID omics, a server for large-scale calculations of protein–RNA interactions. Our web server allows (i) predictions at proteomic and transcriptomic level; (ii) use of protein and RNA sequences without size restriction; (iii) analysis of nucleic acid binding regions in proteins; and (iv) detection of RNA motifs involved in protein recognition.
Results: We developed a web server to allow fast calculation of ribonucleoprotein associations in Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Rattus norvegicus, Saccharomyces cerevisiae and Xenopus tropicalis (custom libraries can be also generated). The catRAPID omics was benchmarked on the recently published RNA interactomes of Serine/arginine-rich splicing factor 1 (SRSF1), Histone-lysine N-methyltransferase EZH2 (EZH2), TAR DNA-binding protein 43 (TDP43) and RNA-binding protein FUS (FUS) as well as on the protein interactomes of U1/U2 small nucleolar RNAs, X inactive specific transcript (Xist) repeat A region (RepA) and Crumbs homolog 3 (CRB3) 3'-untranslated region RNAs. Our predictions are highly significant (P < 0.05) and will help the experimentalist to identify candidates for further validation.
Availability: catRAPID omics can be freely accessed on the Web at http://s.tartaglialab.com/catrapid/omics. Documentation, tutorial and FAQs are available at http://s.tartaglialab.com/page/catrapid_group.
Contact: gian.tartaglia@crg.eu
TALENoffer: genome-wide TALEN off-target prediction [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: Transcription activator-like effector nucleases (TALENs) have become an accepted tool for targeted mutagenesis, but undesired off-targets remain an important issue. We present TALENoffer, a novel tool for the genome-wide prediction of TALEN off-targets. We show that TALENoffer successfully predicts known off-targets of engineered TALENs and yields a competitive runtime, scanning complete mammalian genomes within a few minutes.
Availability: TALENoffer is available as a command line program from http://www.jstacs.de/index.php/TALENoffer and as a Galaxy server at http://galaxy.informatik.uni-halle.de.
Contact: grau@informatik.uni-halle.de
Supplementary Information: Supplementary data are available at Bioinformatics online.
Infernal 1.1: 100-fold faster RNA homology searches [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: Infernal builds probabilistic profiles of the sequence and secondary structure of an RNA family called covariance models (CMs) from structurally annotated multiple sequence alignments given as input. Infernal uses CMs to search for new family members in sequence databases and to create potentially large multiple sequence alignments. Version 1.1 of Infernal introduces a new filter pipeline for RNA homology search based on accelerated profile hidden Markov model (HMM) methods and HMM-banded CM alignment methods. This enables ~100-fold acceleration over the previous version and ~10 000-fold acceleration over exhaustive non-filtered CM searches.
Availability: Source code, documentation and the benchmark are downloadable from http://infernal.janelia.org. Infernal is freely licensed under the GNU GPLv3 and should be portable to any POSIX-compliant operating system, including Linux and Mac OS/X. Documentation includes a user’s guide with a tutorial, a discussion of file formats and user options and additional details on methods implemented in the software.
Contact: nawrockie@janelia.hhmi.org
Consed: a graphical editor for next-generation sequencing [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: The rapid growth of DNA sequencing throughput in recent years implies that graphical interfaces for viewing and correcting errors must now handle large numbers of reads, efficiently pinpoint regions of interest and automate as many tasks as possible. We have adapted consed to reflect this. To allow full-feature editing of large datasets while keeping memory requirements low, we developed a viewer, bamScape, that reads billion-read BAM files, identifies and displays problem areas for user review and launches the consed graphical editor on user-selected regions, allowing, in addition to longstanding consed capabilities such as assembly editing, a variety of new features including direct editing of the reference sequence, variant and error detection, display of annotation tracks and the ability to simultaneously process a group of reads. Many batch processing capabilities have been added.
Availability: The consed package is free to academic, government and non-profit users, and licensed to others for a fee by the University of Washington. The current version (26.0) is available for linux, macosx and solaris systems or as C++ source code. It includes a user’s manual (with exercises) and example datasets. http://www.phrap.org/consed/consed.html
Contact: dgordon@uw.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
RNAfbinv: an interactive Java application for fragment-based design of RNA sequences [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: In RNA design problems, it is plausible to assume that the user would be interested in preserving a particular RNA secondary structure motif, or fragment, for biological reasons. The preservation could be in structure or sequence, or both. Thus, the inverse RNA folding problem could benefit from considering fragment constraints.
We have developed a new interactive Java application called RNA fragment-based inverse that allows users to insert an RNA secondary structure in dot-bracket notation. It then performs sequence design that conforms to the shape of the input secondary structure, the specified thermodynamic stability, the specified mutational robustness and the user-selected fragment after shape decomposition. In this shape-based design approach, specific RNA structural motifs with known biological functions are strictly enforced, while others can possess more flexibility in their structure in favor of preserving physical attributes and additional constraints.
Availability: RNAfbinv is freely available for download on the web at http://www.cs.bgu.ac.il/~RNAexinv/RNAfbinv. The site contains a help file with an explanation regarding the exact use.
Contact: dbarash@cs.bgu.ac.il
RNA secondary structure diagrams for very large molecules: RNAfdl [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: There are many programs that can read the secondary structure of an RNA molecule and draw a diagram, but hardly any that can cope with 103 bases. RNAfdl is slow but capable of producing intersection-free diagrams for ribosome-sized structures, has a graphical user interface for adjustments and produces output in common formats.
Availability and implementation: Source code is available under the GNU General Public License v3.0 at http://sourceforge.net/projects/rnafdl for Linux and similar systems or Windows using MinGW. RNAfdl is implemented in C, uses the Cairo 2D graphics library and offers both command line and graphical user interfaces.
Contact: hecker@rth.dk
Supplementary information: Supplementary data are available at Bioinformatics online.
SAMstrt: statistical test for differential expression in single-cell transcriptome with spike-in normalization [Bioinformatics - recent issues] 2013-10-29 08:48
Motivation: Recent transcriptome studies have revealed that total transcript numbers vary by cell type and condition; therefore, the statistical assumptions for single-cell transcriptome studies must be revisited. SAMstrt is an extension code for SAMseq, which is a statistical method for differential expression, to enable spike-in normalization and statistical testing based on the estimated absolute number of transcripts per cell for single-cell RNA-seq methods.
Availability and Implementation: SAMstrt is implemented on R and available in github (https://github.com/shka/R-SAMstrt).
Contact: shintaro.katayama@ki.se
Supplementary Information: Supplementary data are available at Bioinformatics online.
NetWeAvers: an R package for integrative biological network analysis with mass spectrometry data [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: The discovery of functionally related groups in a set of significantly abundant proteins from a mass spectrometry experiment is an important step in a proteomics analysis pipeline. Here we describe NetWeAvers (Network Weighted Averages) for analyzing groups of regulated proteins in a network context, e.g. as defined by clusters of protein–protein interactions. NetWeAvers is an R package that provides a novel method for analyzing proteomics data integrated with biological networks. The method includes an algorithm for finding dense clusters of proteins and a permutation algorithm to calculate cluster P-values. Optional steps include summarizing quantified peptide values to single protein values and testing for differential expression, such that the data input can simply be a list of identified and quantified peaks.
Availability and implementation: The NetWeAvers package is written in R, is open source and is freely available on CRAN and from netweavers.erasmusmc.nl under the GPL-v2 license.
Contact: e.mcclellan@erasmusmc.nl
Supplementary information: Supplementary data are available at Bioinformatics online.
Metriculator: quality assessment for mass spectrometry-based proteomics [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: Quality control in mass spectrometry-based proteomics remains subjective, labor-intensive and inconsistent between laboratories. We introduce Metriculator, a software designed to facilitate long-term storage of extensive performance metrics as introduced by NIST in 2010. Metriculator features a web interface that generates interactive comparison plots for contextual understanding of metric values and an automated metric generation toolkit. The comparison plots are designed for at-a-glance determination of outliers and trends in the datasets, together with relevant statistical comparisons. Easy-to-use quantitative comparisons and a framework for integration plugins will encourage a culture of quality assurance within the proteomics community.
Availability and Implementation: Available under the MIT license at http://github.com/princelab/metriculator.
Contact: jtprince@chem.byu.edu
The Xeno-glycomics database (XDB): a relational database of qualitative and quantitative pig glycome repertoire [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: In recent years, the improvement of mass spectrometry-based glycomics techniques (i.e. highly sensitive, quantitative and high-throughput analytical tools) has enabled us to obtain a large dataset of glycans. Here we present a database named Xeno-glycomics database (XDB) that contains cell- or tissue-specific pig glycomes analyzed with mass spectrometry-based techniques, including a comprehensive pig glycan information on chemical structures, mass values, types and relative quantities. It was designed as a user-friendly web-based interface that allows users to query the database according to pig tissue/cell types or glycan masses. This database will contribute in providing qualitative and quantitative information on glycomes characterized from various pig cells/organs in xenotransplantation and might eventually provide new targets in the α1,3-galactosyltransferase gene-knock out pigs era.
Availability: The database can be accessed on the web at http://bioinformatics.snu.ac.kr/xdb.
Contact: byungkim@snu.ac.kr or ygkim@ssu.ac.kr
Supplementary information: Supplementary data are available at Bioinformatics online.
M2SG: mapping human disease-related genetic variants to protein sequences and genomic loci [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: Online Mendelian Inheritance in Man (OMIM) is a manually curated compendium of human genetic variants and the corresponding phenotypes, mostly human diseases. Instead of directly documenting the native sequences for gene entries, OMIM links its entries to protein and DNA sequences in other databases. However, because of the existence of gene isoforms and errors in OMIM records, mapping a specific OMIM mutation to its corresponding protein sequence is not trivial. Combining computer programs and extensive manual curation of OMIM full-text descriptions and original literature, we mapped 98% of OMIM amino acid substitutions (AASs) and all SwissProt Variant (SwissVar) disease-related AASs to reference sequences and confidently mapped 99.96% of all AASs to the genomic loci. Based on the results, we developed an online database and interactive web server (M2SG) to (i) retrieve the mapped OMIM and SwissVar variants for a given protein sequence; and (ii) obtain related proteins and mutations for an input disease phenotype. This database will be useful for analyzing sequences, understanding the effect of mutations, identifying important genetic variations and designing experiments on a protein of interest.
Availability and implementation: The database and web server are freely available at http://prodata.swmed.edu/M2S/mut2seq.cgi.
Contact: grishin@chop.swmed.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
OntoQuery: easy-to-use web-based OWL querying [Bioinformatics - recent issues] 2013-10-29 08:48
Summary: The Web Ontology Language (OWL) provides a sophisticated language for building complex domain ontologies and is widely used in bio-ontologies such as the Gene Ontology. The Protégé-OWL ontology editing tool provides a query facility that allows composition and execution of queries with the human-readable Manchester OWL syntax, with syntax checking and entity label lookup. No equivalent query facility such as the Protégé Description Logics (DL) query yet exists in web form. However, many users interact with bio-ontologies such as chemical entities of biological interest and the Gene Ontology using their online Web sites, within which DL-based querying functionality is not available. To address this gap, we introduce the OntoQuery web-based query utility.
Availability and implementation: The source code for this implementation together with instructions for installation is available at http://github.com/IlincaTudose/OntoQuery. OntoQuery software is fully compatible with all OWL-based ontologies and is available for download (CC-0 license). The ChEBI installation, ChEBI OntoQuery, is available at http://www.ebi.ac.uk/chebi/tools/ontoquery.
Contact: hastings@ebi.ac.uk
SubNet: a Java application for subnetwork extraction [Bioinformatics - recent issues] 2013-10-29 08:48
The MaSuRCA genome assembler [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Second-generation sequencing technologies produce high coverage of the genome by short reads at a low cost, which has prompted development of new assembly methods. In particular, multiple algorithms based on de Bruijn graphs have been shown to be effective for the assembly problem. In this article, we describe a new hybrid approach that has the computational efficiency of de Bruijn graph methods and the flexibility of overlap-based assembly strategies, and which allows variable read lengths while tolerating a significant level of sequencing error. Our method transforms large numbers of paired-end reads into a much smaller number of longer ‘super-reads’. The use of super-reads allows us to assemble combinations of Illumina reads of differing lengths together with longer reads from 454 and Sanger sequencing technologies, making it one of the few assemblers capable of handling such mixtures. We call our system the Maryland Super-Read Celera Assembler (abbreviated MaSuRCA and pronounced ‘mazurka’).
Results: We evaluate the performance of MaSuRCA against two of the most widely used assemblers for Illumina data, Allpaths-LG and SOAPdenovo2, on two datasets from organisms for which high-quality assemblies are available: the bacterium Rhodobacter sphaeroides and chromosome 16 of the mouse genome. We show that MaSuRCA performs on par or better than Allpaths-LG and significantly better than SOAPdenovo on these data, when evaluated against the finished sequence. We then show that MaSuRCA can significantly improve its assemblies when the original data are augmented with long reads.
Availability: MaSuRCA is available as open-source code at ftp://ftp.genome.umd.edu/pub/MaSuRCA/. Previous (pre-publication) releases have been publicly available for over a year.
Contact: alekseyz@ipst.umd.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Use of autocorrelation scanning in DNA copy number analysis [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Data quality is a critical issue in the analyses of DNA copy number alterations obtained from microarrays. It is commonly assumed that copy number alteration data can be modeled as piecewise constant and the measurement errors of different probes are independent. However, these assumptions do not always hold in practice. In some published datasets, we find that measurement errors are highly correlated between probes that interrogate nearby genomic loci, and the piecewise-constant model does not fit the data well. The correlated errors cause problems in downstream analysis, leading to a large number of DNA segments falsely identified as having copy number gains and losses.
Method: We developed a simple tool, called autocorrelation scanning profile, to assess the dependence of measurement error between neighboring probes.
Results: Autocorrelation scanning profile can be used to check data quality and refine the analysis of DNA copy number data, which we demonstrate in some typical datasets.
Contact: lzhangli@mdanderson.org
Supplementary information: Supplementary data are available at Bioinformatics online.
PRAP: an ab initio software package for automated genome-wide analysis of DNA repeats for prokaryotes [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Prokaryotic genome annotation has been focused mainly on identifying all genes and their protein functions. However, <30% of the prokaryotic genomes submitted to GenBank contain partial repeat features of specific types and none of the genomes contain complete repeat annotations. Deciphering all repeats in DNA sequences is an important and open task in genome annotation and bioinformatics. Hence, there is an immediate need of a tool capable of identifying full spectrum repeats in the whole genome.
Results: We report the PRAP (Prokaryotic Repeats Annotation Program software package to automate the analysis of repeats in both finished and draft genomes. It is aimed at identifying full spectrum repeats at the scale of the prokaryotic genome. Compared with the major existing repeat finding tools, PRAP exhibits competitive or better results. The results are consistent with manually curated and experimental data. Repeats can be identified and grouped into families to define their relevant types. The final output is parsed into the European Molecular Biology Laboratory (EMBL)/GenBank feature table format for reading and displaying in Artemis, where it can be combined or compared with other genome data. It is currently the most complete repeat finder for prokaryotes and is a valuable tool for genome annotation.
Availability: https://sites.google.com/site/prapsoftware/
Contact: hsuehc@ntu.edu.tw
Supplementary information: Supplementary data are available at Bioinformatics online.
Multiple alignment-free sequence comparison [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Recently, a range of new statistics have become available for the alignment-free comparison of two sequences based on k-tuple word content. Here, we extend these statistics to the simultaneous comparison of more than two sequences. Our suite of statistics contains, first, and , extensions of statistics for pairwise comparison of the joint k-tuple content of all the sequences, and second, , and , averages of sums of pairwise comparison statistics. The two tasks we consider are, first, to identify sequences that are similar to a set of target sequences, and, second, to measure the similarity within a set of sequences.
Results: Our investigation uses both simulated data as well as cis-regulatory module data where the task is to identify cis-regulatory modules with similar transcription factor binding sites. We find that although for real data, all of our statistics show a similar performance, on simulated data the Shepp-type statistics are in some instances outperformed by star-type statistics. The multiple alignment-free statistics are more sensitive to contamination in the data than the pairwise average statistics.
Availability: Our implementation of the five statistics is available as R package named ‘multiAlignFree’ at be http://www-rcf.usc.edu/~fsun/Programs/multiAlignFree/multiAlignFreemain.html.
Contact: reinert@stats.ox.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
A non-independent energy-based multiple sequence alignment improves prediction of transcription factor binding sites [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Multiple sequence alignments (MSAs) are usually scored under the assumption that the sequences being aligned have evolved by common descent. Consequently, the differences between sequences reflect the impact of insertions, deletions and mutations. However, non-coding DNA binding sequences, such as transcription factor binding sites (TFBSs), are frequently not related by common descent, and so the existing alignment scoring methods are not well suited for aligning such sequences.
Results: We present a novel multiple MSA methodology that scores TFBS DNA sequences by including the interdependence of neighboring bases. We introduced two variants supported by different underlying null hypotheses, one statistically and the other thermodynamically generated. We assessed the alignments through their performance in TFBS prediction; both methods show considerable improvements when compared with standard MSA algorithms. Moreover, the thermodynamically generated null hypothesis outperforms the statistical one due to improved stability in the base stacking free energy of the alignment. The thermodynamically generated null hypothesis method can be downloaded from http://sourceforge.net/projects/msa-edna/
Contact: dov.stekel@nottingham.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
Identification of transcription factor binding sites from ChIP-seq data at high resolution [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Chromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) is widely used to study the in vivo binding sites of transcription factors (TFs) and their regulatory targets. Recent improvements to ChIP-seq, such as increased resolution, promise deeper insights into transcriptional regulation, yet require novel computational tools to fully leverage their advantages.
Results: To this aim, we have developed peakzilla, which can identify closely spaced TF binding sites at high resolution (i.e. resolves individual binding sites even if spaced closely), as we demonstrate using semisynthetic datasets, performing ChIP-seq for the TF Twist in Drosophila embryos with different experimental fragment sizes, and analyzing ChIP-exo datasets. We show that the increased resolution reached by peakzilla is highly relevant, as closely spaced Twist binding sites are strongly enriched in transcriptional enhancers, suggesting a signature to discriminate functional from abundant non-functional or neutral TF binding. Peakzilla is easy to use, as it estimates all the necessary parameters from the data and is freely available.
Availability and implementation: The peakzilla program is available from https://github.com/steinmann/peakzilla or http://www.starklab.org/data/peakzilla/.
Contact: stark@starklab.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Accounting for epistatic interactions improves the functional analysis of protein structures [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The constraints under which sequence, structure and function coevolve are not fully understood. Bringing this mutual relationship to light can reveal the molecular basis of binding, catalysis and allostery, thereby identifying function and rationally guiding protein redesign. Underlying these relationships are the epistatic interactions that occur when the consequences of a mutation to a protein are determined by the genetic background in which it occurs. Based on prior data, we hypothesize that epistatic forces operate most strongly between residues nearby in the structure, resulting in smooth evolutionary importance across the structure.
Methods and Results: We find that when residue scores of evolutionary importance are distributed smoothly between nearby residues, functional site prediction accuracy improves. Accordingly, we designed a novel measure of evolutionary importance that focuses on the interaction between pairs of structurally neighboring residues. This measure that we term pair-interaction Evolutionary Trace yields greater functional site overlap and better structure-based proteome-wide functional predictions.
Conclusions: Our data show that the structural smoothness of evolutionary importance is a fundamental feature of the coevolution of sequence, structure and function. Mutations operate on individual residues, but selective pressure depends in part on the extent to which a mutation perturbs interactions with neighboring residues. In practice, this principle led us to redefine the importance of a residue in terms of the importance of its epistatic interactions with neighbors, yielding better annotation of functional residues, motivating experimental validation of a novel functional site in LexA and refining protein function prediction.
Contact: lichtarge@bcm.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
lDDT: a local superposition-free score for comparing protein structures and models using distance difference tests [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The assessment of protein structure prediction techniques requires objective criteria to measure the similarity between a computational model and the experimentally determined reference structure. Conventional similarity measures based on a global superposition of carbon α atoms are strongly influenced by domain motions and do not assess the accuracy of local atomic details in the model.
Results: The Local Distance Difference Test (lDDT) is a superposition-free score that evaluates local distance differences of all atoms in a model, including validation of stereochemical plausibility. The reference can be a single structure, or an ensemble of equivalent structures. We demonstrate that lDDT is well suited to assess local model quality, even in the presence of domain movements, while maintaining good correlation with global measures. These properties make lDDT a robust tool for the automated assessment of structure prediction servers without manual intervention.
Availability and implementation: Source code, binaries for Linux and MacOSX, and an interactive web server are available at http://swissmodel.expasy.org/lddt
Contact: torsten.schwede@unibas.ch
Supplementary information: Supplementary data are available at Bioinformatics online.
Identifying differentially expressed proteins in two-dimensional electrophoresis experiments: inputs from transcriptomics statistical tools [Bioinformatics - recent issues] 2013-10-18 09:02
Background: Two-dimensional electrophoresis is a crucial method in proteomics that allows the characterization of proteins’ function and expression. This usually implies the identification of proteins that are differentially expressed between two contrasting conditions, for example, healthy versus diseased in human proteomics biomarker discovery and stressful conditions versus control in animal experimentation. The statistical procedures that lead to such identifications are critical steps in the 2-DE analysis workflow. They include a normalization step and a test and probability correction for multiple testing. Statistical issues caused by the high dimensionality of the data and large-scale multiple testing have been a more active topic in transcriptomics than proteomics, especially in microarray analysis. We thus propose to adapt innovative statistical tools developed for microarray analysis and incorporate them in the 2-DE analysis pipeline.
Results: In this article, we evaluate the performance of different normalization procedures, different statistical tests and false discovery rate calculation methods with both real and simulated datasets. We demonstrate that the use of statistical procedures adapted from microarrays lead to notable increase in power as well as a minimization of false-positive discovery rate. More specifically, we obtained the best results in terms of reliability and sensibility when using the ‘moderate t-test’ from Smyth in association with classic false discovery rate from Benjamini and Hochberg.
Availability: The methods discussed are freely available in the ‘prot2D’ open source R-package from Bioconductor (http://www.bioconductor.org//) under the terms of the GNU General Public License (version 2 or later).
Contact: sebastien.artigaud@univ-brest.fr or sebastien.artigaud@gmx.com
A combined omics study on activated macrophages--enhanced role of STATs in apoptosis, immunity and lipid metabolism [Bioinformatics - recent issues] 2013-10-18 09:02
Background: Macrophage activation by lipopolysaccharide and adenosine triphosphate (ATP) has been studied extensively because this model system mimics the physiological context of bacterial infection and subsequent inflammatory responses. Previous studies on macrophages elucidated the biological roles of caspase-1 in post-translational activation of interleukin-1β and interleukin-18 in inflammation and apoptosis. However, the results from these studies focused only on a small number of factors. To better understand the host response, we have performed a high-throughput study of Kdo2-lipid A (KLA)-primed macrophages stimulated with ATP.
Results: The study suggests that treating mouse bone marrow-derived macrophages with KLA and ATP produces ‘synergistic’ effects that are not seen with treatment of KLA or ATP alone. The synergistic regulation of genes related to immunity, apoptosis and lipid metabolism is observed in a time-dependent manner. The synergistic effects are produced by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and activator protein (AP)-1 through regulation of their target cytokines. The synergistically regulated cytokines then activate signal transducer and activator of transcription (STAT) factors that result in enhanced immunity, apoptosis and lipid metabolism; STAT1 enhances immunity by promoting anti-microbial factors; and STAT3 contributes to downregulation of cell cycle and upregulation of apoptosis. STAT1 and STAT3 also regulate glycerolipid and eicosanoid metabolism, respectively. Further, western blot analysis for STAT1 and STAT3 showed that the changes in transcriptomic levels were consistent with their proteomic levels. In summary, this study shows the synergistic interaction between the toll-like receptor and purinergic receptor signaling during macrophage activation on bacterial infection.
Availability: Time-course data of transcriptomics and lipidomics can be queried or downloaded from http://www.lipidmaps.org.
Contact: shankar@ucsd.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Imputation of coding variants in African Americans: better performance using data from the exome sequencing project [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: Although the 1000 Genomes haplotypes are the most commonly used reference panel for imputation, medical sequencing projects are generating large alternate sets of sequenced samples. Imputation in African Americans using 3384 haplotypes from the Exome Sequencing Project, compared with 2184 haplotypes from 1000 Genomes Project, increased effective sample size by 8.3–11.4% for coding variants with minor allele frequency <1%. No loss of imputation quality was observed using a panel built from phenotypic extremes. We recommend using haplotypes from Exome Sequencing Project alone or concatenation of the two panels over quality score-based post-imputation selection or IMPUTE2’s two-panel combination.
Contact: yunli@med.unc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Efficient inference of local ancestry [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The inference of local ancestry of admixed individuals at every locus provides the basis for admixture mapping. Local ancestry information has been used to identify genetic susceptibility loci.
Results: In this study, we developed a statistical method, efficient inference of local ancestry (EILA), which uses fused quantile regression and k-means classifier to infer the local ancestry for admixed individuals. We also conducted a simulation study using HapMap data to evaluate the performance of EILA in comparison with two competing methods, HAPMIX and LAMP. In general, the performance declined as the ancestral distance decreased and the time since admixture increased. EILA performed as well as the other two methods in terms of computational efficiency. In the case of closely related ancestral populations, all the three methods performed poorly. Most importantly, when the ancestral distance was large or moderate, EILA had higher accuracy and lower variation in comparison with the other two methods.
Availability and implementation: EILA is implemented as an R package, which is freely available from the Comprehensive R Archive Network (http://cran.r-project.org/).
Contact: jyangstat@gmail.com
Discovering causal pathways linking genomic events to transcriptional states using Tied Diffusion Through Interacting Events (TieDIE) [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Identifying the cellular wiring that connects genomic perturbations to transcriptional changes in cancer is essential to gain a mechanistic understanding of disease initiation, progression and ultimately to predict drug response. We have developed a method called Tied Diffusion Through Interacting Events (TieDIE) that uses a network diffusion approach to connect genomic perturbations to gene expression changes characteristic of cancer subtypes. The method computes a subnetwork of protein–protein interactions, predicted transcription factor-to-target connections and curated interactions from literature that connects genomic and transcriptomic perturbations.
Results: Application of TieDIE to The Cancer Genome Atlas and a breast cancer cell line dataset identified key signaling pathways, with examples impinging on MYC activity. Interlinking genes are predicted to correspond to essential components of cancer signaling and may provide a mechanistic explanation of tumor character and suggest subtype-specific drug targets.
Availability: Software is available from the Stuart lab’s wiki: https://sysbiowiki.soe.ucsc.edu/tiedie.
Contact: jstuart@ucsc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Optimizing a global alignment of protein interaction networks [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The global alignment of protein interaction networks is a widely studied problem. It is an important first step in understanding the relationship between the proteins in different species and identifying functional orthologs. Furthermore, it can provide useful insights into the species’ evolution.
Results: We propose a novel algorithm, PISwap, for optimizing global pairwise alignments of protein interaction networks, based on a local optimization heuristic that has previously demonstrated its effectiveness for a variety of other intractable problems. PISwap can begin with different types of network alignment approaches and then iteratively adjust the initial alignments by incorporating network topology information, trading it off for sequence information. In practice, our algorithm efficiently refines other well-studied alignment techniques with almost no additional time cost. We also show the robustness of the algorithm to noise in protein interaction data. In addition, the flexible nature of this algorithm makes it suitable for different applications of network alignment. This algorithm can yield interesting insights into the evolutionary dynamics of related species.
Availability: Our software is freely available for non-commercial purposes from our Web site, http://piswap.csail.mit.edu/.
Contact: bab@csail.mit.edu or csliao@ie.nthu.edu.tw
Supplementary information: Supplementary data are available at Bioinformatics online.
Multi-profile Bayesian alignment model for LC-MS data analysis with integration of internal standards [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Liquid chromatography-mass spectrometry (LC-MS) has been widely used for profiling expression levels of biomolecules in various ‘-omic’ studies including proteomics, metabolomics and glycomics. Appropriate LC-MS data preprocessing steps are needed to detect true differences between biological groups. Retention time (RT) alignment, which is required to ensure that ion intensity measurements among multiple LC-MS runs are comparable, is one of the most important yet challenging preprocessing steps. Current alignment approaches estimate RT variability using either single chromatograms or detected peaks, but do not simultaneously take into account the complementary information embedded in the entire LC-MS data.
Results: We propose a Bayesian alignment model for LC-MS data analysis. The alignment model provides estimates of the RT variability along with uncertainty measures. The model enables integration of multiple sources of information including internal standards and clustered chromatograms in a mathematically rigorous framework. We apply the model to LC-MS metabolomic, proteomic and glycomic data. The performance of the model is evaluated based on ground-truth data, by measuring correlation of variation, RT difference across runs and peak-matching performance. We demonstrate that Bayesian alignment model improves significantly the RT alignment performance through appropriate integration of relevant information.
Availability and implementation: MATLAB code, raw and preprocessed LC-MS data are available at http://omics.georgetown.edu/alignLCMS.html
Contact: hwr@georgetown.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Exploiting disjointness axioms to improve semantic similarity measures [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Representing domain knowledge in biology has traditionally been accomplished by creating simple hierarchies of classes with textual annotations. Recently, expressive ontology languages, such as Web Ontology Language, have become more widely adopted, supporting axioms that express logical relationships other than class–subclass, e.g. disjointness. This is improving the coverage and validity of the knowledge contained in biological ontologies. However, current semantic tools still need to adapt to this more expressive information. In this article, we propose a method to integrate disjointness axioms, which are being incorporated in real-world ontologies, such as the Gene Ontology and the chemical entities of biological interest ontology, into semantic similarity, the measure that estimates the closeness in meaning between classes.
Results: We present a modification of the measure of shared information content, which extends the base measure to allow the incorporation of disjointness information. To evaluate our approach, we applied it to several randomly selected datasets extracted from the chemical entities of biological interest ontology. In 93.8% of these datasets, our measure performed better than the base measure of shared information content. This supports the idea that semantic similarity is more accurate if it extends beyond the hierarchy of classes of the ontology.
Contact: joao.ferreira@lasige.di.fc.ul.pt
Supplementary information: Supplementary data are available at Bioinformatics online.
Badger--an accessible genome exploration environment [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: High-quality draft genomes are now easy to generate, as sequencing and assembly costs have dropped dramatically. However, building a user-friendly searchable Web site and database for a newly annotated genome is not straightforward. Here we present Badger, a lightweight and easy-to-install genome exploration environment designed for next generation non-model organism genomes.
Availability: Badger is released under the GPL and is available at http://badger.bio.ed.ac.uk/. We show two working examples: (i) a test dataset included with the source code, and (ii) a collection of four filarial nematode genomes.
Contact: mark.blaxter@ed.ac.uk
NextGenMap: fast and accurate read mapping in highly polymorphic genomes [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: When choosing a read mapper, one faces the trade off between speed and the ability to map reads in highly polymorphic regions. Here, we report NextGenMap, a fast and accurate read mapper, which reduces this dilemma. NextGenMap aligns reads reliably to a reference genome even when the sequence difference between target and reference genome is large, i.e. highly polymorphic genome. At the same time, NextGenMap outperforms current mapping methods with respect to runtime and to the number of correctly mapped reads. NextGenMap efficiently uses the available hardware by exploiting multi-core CPUs as well as graphic cards (GPUs), if available. In addition, NextGenMap handles automatically any read data independent of read length and sequencing technology.
Availability: NextGenMap source code and documentation are available at: http://cibiv.github.io/NextGenMap/
Contact: fritz.sedlazeck@univie.ac.at
Supplementary information: Supplementary data are available at Bioinformatics online.
fmcsR: mismatch tolerant maximum common substructure searching in R [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The ability to accurately measure structural similarities among small molecules is important for many analysis routines in drug discovery and chemical genomics. Algorithms used for this purpose include fragment-based fingerprint and graph-based maximum common substructure (MCS) methods. MCS approaches provide one of the most accurate similarity measures. However, their rigid matching policies limit them to the identification of perfect MCSs. To eliminate this restriction, we introduce a new mismatch tolerant search method for identifying flexible MCSs (FMCSs) containing a user-definable number of atom and/or bond mismatches.
Results: The fmcsR package provides an R interface, with the time-consuming steps of the FMCS algorithm implemented in C++. It includes utilities for pairwise compound comparisons, structure similarity searching, clustering and visualization of MCSs. In comparison with an existing MCS tool, fmcsR shows better time performance over a wide range of compound sizes. When mismatching of atoms or bonds is turned on, the compute times increase as expected, and the resulting FMCSs are often substantially larger than their strict MCS counterparts. Based on extensive virtual screening (VS) tests, the flexible matching feature enhances the enrichment of active structures at the top of MCS-based similarity search results. With respect to overall and early enrichment performance, FMCS outperforms most of the seven other VS methods considered in these tests.
Availability: fmcsR is freely available for all common operating systems from the Bioconductor site (http://www.bioconductor.org/packages/devel/bioc/html/fmcsR.html).
Contact: thomas.girke@ucr.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
eSBMTools 1.0: enhanced native structure-based modeling tools [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Molecular dynamics simulations provide detailed insights into the structure and function of biomolecular systems. Thus, they complement experimental measurements by giving access to experimentally inaccessible regimes. Among the different molecular dynamics techniques, native structure-based models (SBMs) are based on energy landscape theory and the principle of minimal frustration. Typically used in protein and RNA folding simulations, they coarse-grain the biomolecular system and/or simplify the Hamiltonian resulting in modest computational requirements while achieving high agreement with experimental data. eSBMTools streamlines running and evaluating SBM in a comprehensive package and offers high flexibility in adding experimental- or bioinformatics-derived restraints.
Results: We present a software package that allows setting up, modifying and evaluating SBM for both RNA and proteins. The implemented workflows include predicting protein complexes based on bioinformatics-derived inter-protein contact information, a standardized setup of protein folding simulations based on the common PDB format, calculating reaction coordinates and evaluating the simulation by free-energy calculations with weighted histogram analysis method or by phi-values. The modules interface with the molecular dynamics simulation program GROMACS. The package is open source and written in architecture-independent Python2.
Availability: http://sourceforge.net/projects/esbmtools/
Contact: alexander.schug@kit.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
iBMQ: a R/Bioconductor package for integrated Bayesian modeling of eQTL data [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: Recently, mapping studies of expression quantitative loci (eQTL) (where gene expression levels are viewed as quantitative traits) have provided insight into the biology of gene regulation. Bayesian methods provide natural modeling frameworks for analyzing eQTL studies, where information shared across markers and/or genes can increase the power to detect eQTLs. Bayesian approaches tend to be computationally demanding and require specialized software. As a result, most eQTL studies use univariate methods treating each gene independently, leading to suboptimal results.
Results: We present a powerful, computationally optimized and free open-source R package, iBMQ. Our package implements a joint hierarchical Bayesian model where all genes and SNPs are modeled concurrently. Model parameters are estimated using a Markov chain Monte Carlo algorithm. The free and widely used openMP parallel library speeds up computation. Using a mouse cardiac dataset, we show that iBMQ improves the detection of large trans-eQTL hotspots compared with other state-of-the-art packages for eQTL analysis.
Availability: The R-package iBMQ is available from the Bioconductor Web site at http://bioconductor.org and runs on Linux, Windows and MAC OS X. It is distributed under the Artistic Licence-2.0 terms.
Contact: christian.deschepper@ircm.qc.ca or rgottard@fhcrc.org
Supplementary information: Supplementary data are available at Bioinformatics online.
ProteoStats--a library for estimating false discovery rates in proteomics pipelines [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: Statistical validation of peptide assignments from a large-scale shotgun proteomics experiment is a critical step, and various methods for evaluating significance based on decoy database search are in practice. False discovery rate (FDR) estimation of peptide assignments assesses global significance and corrects for multiple comparisons. Various approaches have been proposed for FDR estimation but unavailability of standard tools or libraries leads to development of many in-house scripts followed by manual steps that are error-prone and low-throughput. The ProteoStats library provides an open-source framework for developers with many FDR estimation and visualization features for several popular search algorithms. It also provides accurate q-values, which can be easily integrated in any proteomics pipeline to provide automated, accurate, high-throughput statistical validation and minimize manual errors.
Availability: https://sourceforge.net/projects/mssuite/files/ProteoStats/.
Contact: ddash@igib.res.in or aky.compbio@gmail.com or amit.yadav@igib.in
Supplementary information: Supplementary data are available at Bioinformatics online.
Bayesian Network Webserver: a comprehensive tool for biological network modeling [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: The Bayesian Network Webserver (BNW) is a platform for comprehensive network modeling of systems genetics and other biological datasets. It allows users to quickly and seamlessly upload a dataset, learn the structure of the network model that best explains the data and use the model to understand relationships between network variables. Many datasets, including those used to create genetic network models, contain both discrete (e.g. genotype) and continuous (e.g. gene expression traits) variables, and BNW allows for modeling hybrid datasets. Users of BNW can incorporate prior knowledge during structure learning through an easy-to-use structural constraint interface. After structure learning, users are immediately presented with an interactive network model, which can be used to make testable hypotheses about network relationships.
Availability and implementation: BNW, including a downloadable structure learning package, is available at http://compbio.uthsc.edu/BNW. (The BNW interface for adding structural constraints uses HTML5 features that are not supported by current version of Internet Explorer. We suggest using other browsers (e.g. Google Chrome or Mozilla Firefox) when accessing BNW).
Contact: ycui2@uthsc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
LC-IMS-MS Feature Finder: detecting multidimensional liquid chromatography, ion mobility and mass spectrometry features in complex datasets [Bioinformatics - recent issues] 2013-10-18 09:02
Motivation: The addition of ion mobility spectrometry to liquid chromatography-mass spectrometry experiments requires new, or updated, software tools to facilitate data processing.
Results: We introduce a command line software application LC-IMS-MS Feature Finder that searches for molecular ion signatures in multidimensional liquid chromatography-ion mobility spectrometry-mass spectrometry (LC-IMS-MS) data by clustering deisotoped peaks with similar monoisotopic mass, charge state, LC elution time and ion mobility drift time values. The software application includes an algorithm for detecting and quantifying co-eluting chemical species, including species that exist in multiple conformations that may have been separated in the IMS dimension.
Availability: LC-IMS-MS Feature Finder is available as a command-line tool for download at http://omics.pnl.gov/software/LC-IMS-MS_Feature_Finder.php. The Microsoft.NET Framework 4.0 is required to run the software. All other dependencies are included with the software package. Usage of this software is limited to non-profit research to use (see README).
Contact: rds@pnnl.gov
Supplementary information: Supplementary data are available at Bioinformatics online.
HippDB: a database of readily targeted helical protein-protein interactions [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: HippDB catalogs every protein–protein interaction whose structure is available in the Protein Data Bank and which exhibits one or more helices at the interface. The Web site accepts queries on variables such as helix length and sequence, and it provides computational alanine scanning and change in solvent-accessible surface area values for every interfacial residue. HippDB is intended to serve as a starting point for structure-based small molecule and peptidomimetic drug development.
Availability and implementation: HippDB is freely available on the web at http://www.nyu.edu/projects/arora/hippdb. The Web site is implemented in PHP, MySQL and Apache. Source code freely available for download at http://code.google.com/p/helidb, implemented in Perl and supported on Linux.
Contact: arora@nyu.edu
A fast Peptide Match service for UniProt Knowledgebase [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: We have developed a new web application for peptide matching using Apache Lucene-based search engine. The Peptide Match service is designed to quickly retrieve all occurrences of a given query peptide from UniProt Knowledgebase (UniProtKB) with isoforms. The matched proteins are shown in summary tables with rich annotations, including matched sequence region(s) and links to corresponding proteins in a number of proteomic/peptide spectral databases. The results are grouped by taxonomy and can be browsed by organism, taxonomic group or taxonomy tree. The service supports queries where isobaric leucine and isoleucine are treated equivalent, and an option for searching UniRef100 representative sequences, as well as dynamic queries to major proteomic databases. In addition to the web interface, we also provide RESTful web services. The underlying data are updated every 4 weeks in accordance with the UniProt releases.
Availability: http://proteininformationresource.org/peptide.shtml
Contact: chenc@udel.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
RDAVIDWebService: a versatile R interface to DAVID [Bioinformatics - recent issues] 2013-10-18 09:02
Summary: The RDAVIDWebService package provides a class-based interface from R programs/scripts to fully access/control the database for annotation, visualization and integrated discovery, without the need for human interaction on its Web site (http://david.abcc.ncifcrf.gov). The library enhances the database for annotation, visualization and integrated discovery capabilities for Gene Ontology analysis by means of GOstats-based direct acyclic graph conversion methods, in addition to the usual many-genes-to-many-terms visualization.
Availability and implementation: RDAVIDWebService is available as an R package from the Bioconductor project (www.bioconductor.org) and on the authors’ Web site (www.bdmg.com.ar) under GPL-2 license, subjected to the terms of use of DAVID (http://david.abcc.ncifcrf.gov/content.jsp?file=WS.html).
isomiRID: a framework to identify microRNA isoforms [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: MicroRNAs (miRNAs) have been extensively studied owing to their important regulatory roles in genic expression. An increasingly number of reports are performing extensive data mining in small RNA sequencing libraries to detect miRNAs isoforms and also 5' and 3' post-transcriptional nucleotide additions, as well as edited miRNAs sequences. A ready to use pipeline, isomiRID, was developed to standardize and automatize the search for miRNAs isoforms in high-throughput small RNA sequencing libraries.
Availability: isomiRID is a command line Python script available at http://www.ufrgs.br/RNAi/isomiRID/.
Contact: rogerio.margis@ufrgs.br
Supplementary information: Supplementary Date are available at Bioinformatics online.
Analysis of base-pairing probabilities of RNA molecules involved in protein-RNA interactions [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Understanding the details of protein–RNA interactions is important to reveal the functions of both the RNAs and the proteins. In these interactions, the secondary structures of the RNAs play an important role. Because RNA secondary structures in protein–RNA complexes are variable, considering the ensemble of RNA secondary structures is a useful approach. In particular, recent studies have supported the idea that, in the analysis of RNA secondary structures, the base-pairing probabilities (BPPs) of RNAs (i.e. the probabilities of forming a base pair in the ensemble of RNA secondary structures) provide richer and more robust information about the structures than a single RNA secondary structure, for example, the minimum free energy structure or a snapshot of structures in the Protein Data Bank. However, there has been no investigation of the BPPs in protein–RNA interactions.
Results: In this study, we analyzed BPPs of RNA molecules involved in known protein–RNA complexes in the Protein Data Bank. Our analysis suggests that, in the tertiary structures, the BPPs (which are computed using only sequence information) for unpaired nucleotides with intermolecular hydrogen bonds (hbonds) to amino acids were significantly lower than those for unpaired nucleotides without hbonds. On the other hand, no difference was found between the BPPs for paired nucleotides with and without intermolecular hbonds. Those findings were commonly supported by three probabilistic models, which provide the ensemble of RNA secondary structures, including the McCaskill model based on Turner’s free energy of secondary structures.
Contact: iwakiri@cb.k.u-tokyo.ac.jp or mhamada@cb.k.u-tokyo.ac.jp
Supplementary information: Supplementary data are available at Bioinformatics online.
MITIE: Simultaneous RNA-Seq-based transcript identification and quantification in multiple samples [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: High-throughput sequencing of mRNA (RNA-Seq) has led to tremendous improvements in the detection of expressed genes and reconstruction of RNA transcripts. However, the extensive dynamic range of gene expression, technical limitations and biases, as well as the observed complexity of the transcriptional landscape, pose profound computational challenges for transcriptome reconstruction.
Results: We present the novel framework MITIE (Mixed Integer Transcript IdEntification) for simultaneous transcript reconstruction and quantification. We define a likelihood function based on the negative binomial distribution, use a regularization approach to select a few transcripts collectively explaining the observed read data and show how to find the optimal solution using Mixed Integer Programming. MITIE can (i) take advantage of known transcripts, (ii) reconstruct and quantify transcripts simultaneously in multiple samples, and (iii) resolve the location of multi-mapping reads. It is designed for genome- and assembly-based transcriptome reconstruction. We present an extensive study based on realistic simulated RNA-Seq data. When compared with state-of-the-art approaches, MITIE proves to be significantly more sensitive and overall more accurate. Moreover, MITIE yields substantial performance gains when used with multiple samples. We applied our system to 38 Drosophila melanogaster modENCODE RNA-Seq libraries and estimated the sensitivity of reconstructing omitted transcript annotations and the specificity with respect to annotated transcripts. Our results corroborate that a well-motivated objective paired with appropriate optimization techniques lead to significant improvements over the state-of-the-art in transcriptome reconstruction.
Availability: MITIE is implemented in C++ and is available from http://bioweb.me/mitie under the GPL license.
Contact: Jonas_Behr@web.de and raetsch@cbio.mskcc.org
Supplementary information: Supplementary data are available at Bioinformatics online.
Oncofuse: a computational framework for the prediction of the oncogenic potential of gene fusions [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Gene fusions resulting from chromosomal aberrations are an important cause of cancer. The complexity of genomic changes in certain cancer types has hampered the identification of gene fusions by molecular cytogenetic methods, especially in carcinomas. This is changing with the advent of next-generation sequencing, which is detecting a substantial number of new fusion transcripts in individual cancer genomes. However, this poses the challenge of identifying those fusions with greater oncogenic potential amid a background of ‘passenger’ fusion sequences.
Results: In the present work, we have used some recently identified genomic hallmarks of oncogenic fusion genes to develop a pipeline for the classification of fusion sequences, namely, Oncofuse. The pipeline predicts the oncogenic potential of novel fusion genes, calculating the probability that a fusion sequence behaves as ‘driver’ of the oncogenic process based on features present in known oncogenic fusions. Cross-validation and extensive validation tests on independent datasets suggest a robust behavior with good precision and recall rates. We believe that Oncofuse could become a useful tool to guide experimental validation studies of novel fusion sequences found during next-generation sequencing analysis of cancer transcriptomes.
Availability and implementation: Oncofuse is a naive Bayes Network Classifier trained and tested using Weka machine learning package. The pipeline is executed by running a Java/Groovy script, available for download at www.unav.es/genetica/oncofuse.html.
Contact: fnovo@unav.es
Supplementary information: Supplementary data are available at Bioinformatics online.
Inferring nucleosome positions with their histone mark annotation from ChIP data [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: The nucleosome is the basic repeating unit of chromatin. It contains two copies each of the four core histones H2A, H2B, H3 and H4 and about 147 bp of DNA. The residues of the histone proteins are subject to numerous post-translational modifications, such as methylation or acetylation. Chromatin immunoprecipitiation followed by sequencing (ChIP-seq) is a technique that provides genome-wide occupancy data of these modified histone proteins, and it requires appropriate computational methods.
Results: We present NucHunter, an algorithm that uses the data from ChIP-seq experiments directed against many histone modifications to infer positioned nucleosomes. NucHunter annotates each of these nucleosomes with the intensities of the histone modifications. We demonstrate that these annotations can be used to infer nucleosomal states with distinct correlations to underlying genomic features and chromatin-related processes, such as transcriptional start sites, enhancers, elongation by RNA polymerase II and chromatin-mediated repression. Thus, NucHunter is a versatile tool that can be used to predict positioned nucleosomes from a panel of histone modification ChIP-seq experiments and infer distinct histone modification patterns associated to different chromatin states.
Availability: The software is available at http://epigen.molgen.mpg.de/nuchunter/.
Contact: chung@molgen.mpg.de
Supplementary information: Supplementary data are available at Bioinformatics online.
A distance-based test of association between paired heterogeneous genomic data [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Due to rapid technological advances, a wide range of different measurements can be obtained from a given biological sample including single nucleotide polymorphisms, copy number variation, gene expression levels, DNA methylation and proteomic profiles. Each of these distinct measurements provides the means to characterize a certain aspect of biological diversity, and a fundamental problem of broad interest concerns the discovery of shared patterns of variation across different data types. Such data types are heterogeneous in the sense that they represent measurements taken at different scales or represented by different data structures.
Results: We propose a distance-based statistical test, the generalized RV (GRV) test, to assess whether there is a common and non-random pattern of variability between paired biological measurements obtained from the same random sample. The measurements enter the test through the use of two distance measures, which can be chosen to capture a particular aspect of the data. An approximate null distribution is proposed to compute P-values in closed-form and without the need to perform costly Monte Carlo permutation procedures. Compared with the classical Mantel test for association between distance matrices, the GRV test has been found to be more powerful in a number of simulation settings. We also demonstrate how the GRV test can be used to detect biological pathways in which genetic variability is associated to variation in gene expression levels in an ovarian cancer sample, and present results obtained from two independent cohorts.
Availability: R code to compute the GRV test is freely available from http://www2.imperial.ac.uk/~gmontana
Contact: g.montana@imperial.ac.uk
Supplementary data: Supplementary data are available at Bioinformatics online.
Accurate identification of polyadenylation sites from 3' end deep sequencing using a naive Bayes classifier [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: 3' end processing is important for transcription termination, mRNA stability and regulation of gene expression. To identify 3' ends, most techniques use an oligo-dT primer to construct deep sequencing libraries. However, this approach can lead to identification of artifactual polyadenylation sites due to internal priming in homopolymeric stretches of adenines. Although heuristic filters have been applied in these cases, they typically result in a high proportion of both false-positive and -negative classifications. Therefore, there is a need to develop improved algorithms to better identify mis-priming events in oligo-dT primed sequences.
Results: By analyzing sequence features flanking 3' ends derived from oligo-dT-based sequencing, we developed a naïve Bayes classifier to classify them as true or false/internally primed. The resulting algorithm is highly accurate, outperforms previous heuristic filters and facilitates identification of novel polyadenylation sites.
Contact: nathan.lawson@umassmed.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Genome compression: a novel approach for large collections [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Genomic repositories are rapidly growing, as witnessed by the 1000 Genomes or the UK10K projects. Hence, compression of multiple genomes of the same species has become an active research area in the past years. The well-known large redundancy in human sequences is not easy to exploit because of huge memory requirements from traditional compression algorithms.
Results: We show how to obtain several times higher compression ratio than of the best reported results, on two large genome collections (1092 human and 775 plant genomes). Our inputs are variant call format files restricted to their essential fields. More precisely, our novel Ziv-Lempel-style compression algorithm squeezes a single human genome to ~400 KB. The key to high compression is to look for similarities across the whole collection, not just against one reference sequence, what is typical for existing solutions.
Availability: http://sun.aei.polsl.pl/tgc (also as Supplementary Material) under a free license.
Supplementary data: Supplementary data are available at Bioinformatics online.
Contact: sebastian.deorowicz@polsl.pl
High-accuracy prediction of transmembrane inter-helix contacts and application to GPCR 3D structure modeling [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Residue–residue contacts across the transmembrane helices dictate the three-dimensional topology of alpha-helical membrane proteins. However, contact determination through experiments is difficult because most transmembrane proteins are hard to crystallize.
Results: We present a novel method (MemBrain) to derive transmembrane inter-helix contacts from amino acid sequences by combining correlated mutations and multiple machine learning classifiers. Tested on 60 non-redundant polytopic proteins using a strict leave-one-out cross-validation protocol, MemBrain achieves an average accuracy of 62%, which is 12.5% higher than the current best method from the literature. When applied to 13 recently solved G protein-coupled receptors, the MemBrain contact predictions helped increase the TM-score of the I-TASSER models by 37% in the transmembrane region. The number of foldable cases (TM-score >0.5) increased by 100%, where all G protein-coupled receptor templates and homologous templates with sequence identity >30% were excluded. These results demonstrate significant progress in contact prediction and a potential for contact-driven structure modeling of transmembrane proteins.
Availability: www.csbio.sjtu.edu.cn/bioinf/MemBrain/
Contact: hbshen@sjtu.edu.cn or zhng@umich.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Identification of protein–ligand binding sites is critical to protein function annotation and drug discovery. However, there is no method that could generate optimal binding site prediction for different protein types. Combination of complementary predictions is probably the most reliable solution to the problem.
Results: We develop two new methods, one based on binding-specific substructure comparison (TM-SITE) and another on sequence profile alignment (S-SITE), for complementary binding site predictions. The methods are tested on a set of 500 non-redundant proteins harboring 814 natural, drug-like and metal ion molecules. Starting from low-resolution protein structure predictions, the methods successfully recognize >51% of binding residues with average Matthews correlation coefficient (MCC) significantly higher (with P-value <10–9 in student t-test) than other state-of-the-art methods, including COFACTOR, FINDSITE and ConCavity. When combining TM-SITE and S-SITE with other structure-based programs, a consensus approach (COACH) can increase MCC by 15% over the best individual predictions. COACH was examined in the recent community-wide COMEO experiment and consistently ranked as the best method in last 22 individual datasets with the Area Under the Curve score 22.5% higher than the second best method. These data demonstrate a new robust approach to protein–ligand binding site recognition, which is ready for genome-wide structure-based function annotations.
Availability: http://zhanglab.ccmb.med.umich.edu/COACH/
Contact: zhng@umich.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Identification of active transcription factor and miRNA regulatory pathways in Alzheimer's disease [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Alzheimer’s disease (AD) is a severe neurodegenerative disease of the central nervous system that may be caused by perturbation of regulatory pathways rather than the dysfunction of a single gene. However, the pathology of AD has yet to be fully elucidated.
Results: In this study, we systematically analyzed AD-related mRNA and miRNA expression profiles as well as curated transcription factor (TF) and miRNA regulation to identify active TF and miRNA regulatory pathways in AD. By mapping differentially expressed genes and miRNAs to the curated TF and miRNA regulatory network as active seed nodes, we obtained a potential active subnetwork in AD. Next, by using the breadth-first-search technique, potential active regulatory pathways, which are the regulatory cascade of TFs, miRNAs and their target genes, were identified. Finally, based on the known AD-related genes and miRNAs, the hypergeometric test was used to identify active pathways in AD. As a result, nine pathways were found to be significantly activated in AD. A comprehensive literature review revealed that eight out of nine genes and miRNAs in these active pathways were associated with AD. In addition, we inferred that the pathway hsa-miR-146a->STAT1->MYC, which is the source of all nine significantly active pathways, may play an important role in AD progression, which should be further validated by biological experiments. Thus, this study provides an effective approach to finding active TF and miRNA regulatory pathways in AD and can be easily applied to other complex diseases.
Contact: lixia@hrbmu.edu.cn or lw2247@gmail.com.
Supplementary information: Supplementary data are available at Bioinformatics online.
A Turing test for artificial expression data [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: The lack of reliable, comprehensive gold standards complicates the development of many bioinformatics tools, particularly for the analysis of expression data and biological networks. Simulation approaches can provide provisional gold standards, such as regulatory networks, for the assessment of network inference methods. However, this just defers the problem, as it is difficult to assess how closely simulators emulate the properties of real data.
Results: In analogy to Turing’s test discriminating humans and computers based on responses to questions, we systematically compare real and artificial systems based on their gene expression output. Different expression data analysis techniques such as clustering are applied to both types of datasets. We define and extract distributions of properties from the results, for instance, distributions of cluster quality measures or transcription factor activity patterns. Distributions of properties are represented as histograms to enable the comparison of artificial and real datasets. We examine three frequently used simulators that generate expression data from parameterized regulatory networks. We identify features distinguishing real from artificial datasets that suggest how simulators could be adapted to better emulate real datasets and, thus, become more suitable for the evaluation of data analysis tools.
Contact: robert.kueffner@bio.ifi.lmu.de
Supplementary information: Supplementary data are available at Bioinformatics online.
Bayesian consensus clustering [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: In biomedical research a growing number of platforms and technologies are used to measure diverse but related information, and the task of clustering a set of objects based on multiple sources of data arises in several applications. Most current approaches to multisource clustering either independently determine a separate clustering for each data source or determine a single ‘joint’ clustering for all data sources. There is a need for more flexible approaches that simultaneously model the dependence and the heterogeneity of the data sources.
Results: We propose an integrative statistical model that permits a separate clustering of the objects for each data source. These separate clusterings adhere loosely to an overall consensus clustering, and hence they are not independent. We describe a computationally scalable Bayesian framework for simultaneous estimation of both the consensus clustering and the source-specific clusterings. We demonstrate that this flexible approach is more robust than joint clustering of all data sources, and is more powerful than clustering each data source independently. We present an application to subtype identification of breast cancer tumor samples using publicly available data from The Cancer Genome Atlas.
Availability: R code with instructions and examples is available at http://people.duke.edu/%7Eel113/software.html.
Contact: Eric.Lock@duke.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
Novel human lncRNA-disease association inference based on lncRNA expression profiles [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: More and more evidences have indicated that long–non-coding RNAs (lncRNAs) play critical roles in many important biological processes. Therefore, mutations and dysregulations of these lncRNAs would contribute to the development of various complex diseases. Developing powerful computational models for potential disease-related lncRNAs identification would benefit biomarker identification and drug discovery for human disease diagnosis, treatment, prognosis and prevention.
Results: In this article, we proposed the assumption that similar diseases tend to be associated with functionally similar lncRNAs. Then, we further developed the method of Laplacian Regularized Least Squares for LncRNA–Disease Association (LRLSLDA) in the semisupervised learning framework. Although known disease–lncRNA associations in the database are rare, LRLSLDA still obtained an AUC of 0.7760 in the leave-one-out cross validation, significantly improving the performance of previous methods. We also illustrated the performance of LRLSLDA is not sensitive (even robust) to the parameters selection and it can obtain a reliable performance in all the test classes. Plenty of potential disease–lncRNA associations were publicly released and some of them have been confirmed by recent results in biological experiments. It is anticipated that LRLSLDA could be an effective and important biological tool for biomedical research.
Availability: The code of LRLSLDA is freely available at http://asdcd.amss.ac.cn/Software/Details/2.
Contact: xingchen@amss.ac.cn or yangy@amt.ac.cn
Supplementary information: Supplementary data are available at Bioinformatics online.
Near-optimal experimental design for model selection in systems biology [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: Biological systems are understood through iterations of modeling and experimentation. Not all experiments, however, are equally valuable for predictive modeling. This study introduces an efficient method for experimental design aimed at selecting dynamical models from data. Motivated by biological applications, the method enables the design of crucial experiments: it determines a highly informative selection of measurement readouts and time points.
Results: We demonstrate formal guarantees of design efficiency on the basis of previous results. By reducing our task to the setting of graphical models, we prove that the method finds a near-optimal design selection with a polynomial number of evaluations. Moreover, the method exhibits the best polynomial-complexity constant approximation factor, unless P = NP. We measure the performance of the method in comparison with established alternatives, such as ensemble non-centrality, on example models of different complexity. Efficient design accelerates the loop between modeling and experimentation: it enables the inference of complex mechanisms, such as those controlling central metabolic operation.
Availability: Toolbox ‘NearOED’ available with source code under GPL on the Machine Learning Open Source Software Web site (mloss.org).
Contact: busettoa@inf.ethz.ch
Supplementary information: Supplementary data are available at Bioinformatics online.
Incorporating prior knowledge into Gene Network Study [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: A major goal in genomic research is to identify genes that may jointly influence a biological response. From many years of intensive biomedical research, a large body of biological knowledge, or pathway information, has accumulated in available databases. There is a strong interest in leveraging these pathways to improve the statistical power and interpretability in studying gene networks associated with complex phenotypes. This prior information is a valuable complement to large-scale genomic data such as gene expression data generated from microarrays. However, it is a non-trivial task to effectively integrate available biological knowledge into gene expression data when reconstructing gene networks.
Results: In this article, we developed and applied a Lasso method from a Bayesian perspective, a method we call prior Lasso (pLasso), for the reconstruction of gene networks. In this method, we partition edges between genes into two subsets: one subset of edges is present in known pathways, whereas the other has no prior information associated. Our method assigns different prior distributions to each subset according to a modified Bayesian information criterion that incorporates prior knowledge on both the network structure and the pathway information. Simulation studies have indicated that the method is more effective in recovering the underlying network than a traditional Lasso method that does not use the prior information. We applied pLasso to microarray gene expression datasets, where we used information from the Pathway Commons (PC) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) as prior information for the network reconstruction, and successfully identified network hub genes associated with clinical outcome in cancer patients.
Availability: The source code is available at http://nba.uth.tmc.edu/homepage/liu/pLasso.
Contact: Yin.Liu@uth.tmc.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
SPOCS: software for predicting and visualizing orthology/paralogy relationships among genomes [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: At the rate that prokaryotic genomes can now be generated, comparative genomics studies require a flexible method for quickly and accurately predicting orthologs among the rapidly changing set of genomes available. SPOCS implements a graph-based ortholog prediction method to generate a simple tab-delimited table of orthologs and in addition, html files that provide a visualization of the predicted ortholog/paralog relationships to which gene/protein expression metadata may be overlaid.
Availability and Implementation: A SPOCS web application is freely available at http://cbb.pnnl.gov/portal/tools/spocs.html. Source code for Linux systems is also freely available under an open source license at http://cbb.pnnl.gov/portal/software/spocs.html; the Boost C++ libraries and BLAST are required.
Contact: leeann.mccue@pnnl.gov
PARSEC: PAtteRn SEarch and Contextualization [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: We present PARSEC (PAtteRn Search and Contextualization), a new open source platform for guided discovery, allowing localization and biological characterization of short genomic sites in entire eukaryotic genomes. PARSEC can search for a sequence or a degenerated pattern. The retrieved set of genomic sites can be characterized in terms of (i) conservation in model organisms, (ii) genomic context (proximity to genes) and (iii) function of neighboring genes. These modules allow the user to explore, visualize, filter and extract biological knowledge from a set of short genomic regions such as transcription factor binding sites.
Availability: Web site implemented in Java, JavaScript and C++, with all major browsers supported. Freely available at lbgi.fr/parsec. Source code is freely available at sourceforge.net/projects/genomicparsec.
Contact: odile.lecompte@unistra.fr
Supplementary information: Supplementary data are available at Bioinformatics online.
MLML: consistent simultaneous estimates of DNA methylation and hydroxymethylation [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: The two major epigenetic modifications of cytosines, 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC), coexist with each other in a range of mammalian cell populations. Increasing evidence points to important roles of 5-hmC in demethylation of 5-mC and epigenomic regulation in development. Recently developed experimental methods allow direct single-base profiling of either 5-hmC or 5-mC. Meaningful analyses seem to require combining these experiments with bisulfite sequencing, but doing so naively produces inconsistent estimates of 5-mC or 5-hmC levels.
Results: We present a method to jointly model read counts from bisulfite sequencing, oxidative bisulfite sequencing and Tet-Assisted Bisulfite sequencing, providing simultaneous estimates of 5-hmC and 5-mC levels that are consistent across experiment types.
Availability: http://smithlab.usc.edu/software/mlml
Contact: andrewds@usc.edu
Supplementary information: Supplementary material is available at Bioinformatics online.
Pclust: protein network visualization highlighting experimental data [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: One approach to infer functions of new proteins from their homologs utilizes visualization of an all-against-all pairwise similarity network (A2ApsN) that exploits the speed of BLAST and avoids the complexity of multiple sequence alignment. However, identifying functions of the protein clusters in A2ApsN is never trivial, due to a lack of linking characterized proteins to their relevant information in current software packages. Given the database errors introduced by automatic annotation transfer, functional deduction should be made from proteins with experimental studies, i.e. ‘reference proteins’. Here, we present a web server, termed Pclust, which provides a user-friendly interface to visualize the A2ApsN, placing emphasis on such ‘reference proteins’ and providing access to their full information in source databases, e.g. articles in PubMed. The identification of ‘reference proteins’ and the ease of cross-database linkage will facilitate understanding the functions of protein clusters in the network, thus promoting interpretation of proteins of interest.
Availability: The Pclust server is freely available at http://prodata.swmed.edu/pclust
Contact: grishin@chop.swmed.edu
Supplementary Information: Supplementary data are available at Bioinformatics online.
WebRASP: a server for computing energy scores to assess the accuracy and stability of RNA 3D structures [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: The understanding of the biological role of RNA molecules has changed. Although it is widely accepted that RNAs play important regulatory roles without necessarily coding for proteins, the functions of many of these non-coding RNAs are unknown. Thus, determining or modeling the 3D structure of RNA molecules as well as assessing their accuracy and stability has become of great importance for characterizing their functional activity. Here, we introduce a new web application, WebRASP, that uses knowledge-based potentials for scoring RNA structures based on distance-dependent pairwise atomic interactions. This web server allows the users to upload a structure in PDB format, select several options to visualize the structure and calculate the energy profile. The server contains online help, tutorials and links to other related resources. We believe this server will be a useful tool for predicting and assessing the quality of RNA 3D structures.
Availability and implementation: The web server is available at http://melolab.org/webrasp. It has been tested on the most popular web browsers and requires Java plugin for Jmol visualization.
Contact: fmelo@bio.puc.cl
omiRas: a Web server for differential expression analysis of miRNAs derived from small RNA-Seq data [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: Small RNA deep sequencing is widely used to characterize non-coding RNAs (ncRNAs) differentially expressed between two conditions, e.g. healthy and diseased individuals and to reveal insights into molecular mechanisms underlying condition-specific phenotypic traits. The ncRNAome is composed of a multitude of RNAs, such as transfer RNA, small nucleolar RNA and microRNA (miRNA), to name few. Here we present omiRas, a Web server for the annotation, comparison and visualization of interaction networks of ncRNAs derived from next-generation sequencing experiments of two different conditions. The Web tool allows the user to submit raw sequencing data and results are presented as: (i) static annotation results including length distribution, mapping statistics, alignments and quantification tables for each library as well as lists of differentially expressed ncRNAs between conditions and (ii) an interactive network visualization of user-selected miRNAs and their target genes based on the combination of several miRNA–mRNA interaction databases.
Availability and Implementation: The omiRas Web server is implemented in Python, PostgreSQL, R and can be accessed at: http://tools.genxpro.net/omiras/.
Contact: rotter@genxpro.de
Supplementary Information: Supplementary data are available at Bioinformatics online.
nCal: an R package for non-linear calibration [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: Non-linear calibration is a widely used method for quantifying biomarkers wherein concentration-response curves estimated using samples of known concentrations are used to predict the biomarker concentrations in the samples of interest. The R package nCal fills an important gap in the open source, stand-alone software for performing non-linear calibration. For curve fitting, nCal provides a new implementation of a robust, Bayesian hierarchical five-parameter logistic model. nCal supports a simple graphical user interface that can be used by laboratory scientists, and contains functionality for importing data from the multiplex bead array assay instrumentation.
Availability: The R package ‘nCal’ is available from http://cran.r-project.org/web/packages/nCal/ under GPL-2 or later.
Contact: yfong@fhcrc.org
Supplementary information: Supplementary information is available in the form of an R package vignette at the above repository and an FAQ at http://research.fhcrc.org/youyifong/en/resources/ncal.html.
Scaffold network generator: a tool for mining molecular structures [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: Scaffold network generator (SNG) is an open-source command-line utility that computes the hierarchical network of scaffolds that define a large set of input molecules. Scaffold networks are useful for visualizing, analysing and understanding the chemical data that is increasingly available through large public repositories like PubChem. For example, some groups have used scaffold networks to identify missed-actives in high-throughput screens of small molecules with bioassays. Substantially improving on existing software, SNG is robust enough to work on millions of molecules at a time with a simple command-line interface.
Availability and implementation: SNG is accessible at http://swami.wustl.edu/sng
Contact: swamidass@wustl.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
targetHub: a programmable interface for miRNA-gene interactions [Bioinformatics - recent issues] 2013-10-03 09:49
Motivation: With the expansion of high-throughput technologies, understanding different kinds of genome-level data is a common task. MicroRNA (miRNA) is increasingly profiled using high-throughput technologies (microarrays or next-generation sequencing). The downstream analysis of miRNA targets can be difficult. Although there are many databases and algorithms to predict miRNA targets, there are few tools to integrate miRNA–gene interaction data into high-throughput genomic analyses.
Results: We present targetHub, a CouchDB database of miRNA–gene interactions. TargetHub provides a programmer-friendly interface to access miRNA targets. The Web site provides RESTful access to miRNA–gene interactions with an assortment of gene and miRNA identifiers. It can be a useful tool to integrate miRNA target interaction data directly into high-throughput bioinformatics analyses.
Availability: TargetHub is available on the web at http://app1.bioinformatics.mdanderson.org/tarhub/_design/basic/index.html.
Contact: coombes.3@osu.edu
The BioPAX Validator [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: BioPAX is a community-developed standard language for biological pathway data. A key functionality required for efficient BioPAX data exchange is validation—detecting errors and inconsistencies in BioPAX documents. The BioPAX Validator is a command-line tool, Java library and online web service for BioPAX that performs >100 classes of consistency checks.
Availability and implementation: The validator recognizes common syntactic errors and semantic inconsistencies and reports them in a customizable human readable format. It can also automatically fix some errors and normalize BioPAX data. Since its release, the validator has become a critical tool for the pathway informatics community, detecting thousands of errors and helping substantially increase the conformity and uniformity of BioPAX-formatted data. The BioPAX Validator is open source and released under LGPL v3 license. All sources, binaries and documentation can be found at sf.net/p/biopax, and the latest stable version of the web application is available at biopax.org/validator.
Contact: igor.rodchenkov@utoronto.ca or gary.bader@utoronto.ca
CellMissy: a tool for management, storage and analysis of cell migration data produced in wound healing-like assays [Bioinformatics - recent issues] 2013-10-03 09:49
Summary: Automated image processing has allowed cell migration research to evolve to a high-throughput research field. As a consequence, there is now an unmet need for data management in this domain. The absence of a generic management system for the quantitative data generated in cell migration assays results in each dataset being treated in isolation, making data comparison across experiments difficult. Moreover, by integrating quality control and analysis capabilities into such a data management system, the common practice of having to manually transfer data across different downstream analysis tools will be markedly sped up and made more robust. In addition, access to a data management solution creates gateways for data standardization, meta-analysis and structured public data dissemination.
We here present CellMissy, a cross-platform data management system for cell migration data with a focus on wound healing data. CellMissy simplifies and automates data management, storage and analysis from the initial experimental set-up to data exploration.
Availability and implementation: CellMissy is a cross-platform open-source software developed in Java. Source code and cross-platform binaries are freely available under the Apache2 open source license at http://cellmissy.googlecode.com.
Contact: lennart.martens@ugent.be
Supplementary Information: Supplementary data are available at Bioinformatics online.
Engineered proteins detect spontaneous DNA breakage in human and bacterial cells [eLife recent issues] 2013-10-29 10:54
Spontaneous DNA breaks instigate genomic changes that fuel cancer and evolution, yet direct quantification of double-strand breaks (DSBs) has been limited. Predominant sources of spontaneous DSBs remain elusive. We report synthetic technology for quantifying DSBs using fluorescent-protein fusions of double-strand DNA end-binding protein, Gam of bacteriophage Mu. In Escherichia coli GamGFP forms foci at chromosomal DSBs and pinpoints their subgenomic locations. Spontaneous DSBs occur mostly one per cell, and correspond with generations, supporting replicative models for spontaneous breakage, and providing the first true breakage rates. In mammalian cells GamGFP—labels laser-induced DSBs antagonized by end-binding protein Ku; co-localizes incompletely with DSB marker 53BP1 suggesting superior DSB-specificity; blocks resection; and demonstrates DNA breakage via APOBEC3A cytosine deaminase. We demonstrate directly that some spontaneous DSBs occur outside of S phase. The data illuminate spontaneous DNA breakage in E. coli and human cells and illustrate the versatility of fluorescent-Gam for interrogation of DSBs in living cells.
Global cellular response to chemotherapy-induced apoptosis [eLife recent issues] 2013-10-29 10:54
How cancer cells globally struggle with a chemotherapeutic insult before succumbing to apoptosis is largely unknown. Here we use an integrated systems-level examination of transcription, translation, and proteolysis to understand these events central to cancer treatment. As a model we study myeloma cells exposed to the proteasome inhibitor bortezomib, a first-line therapy. Despite robust transcriptional changes, unbiased quantitative proteomics detects production of only a few critical anti-apoptotic proteins against a background of general translation inhibition. Simultaneous ribosome profiling further reveals potential translational regulation of stress response genes. Once the apoptotic machinery is engaged, degradation by caspases is largely independent of upstream bortezomib effects. Moreover, previously uncharacterized non-caspase proteolytic events also participate in cellular deconstruction. Our systems-level data also support co-targeting the anti-apoptotic regulator HSF1 to promote cell death by bortezomib. This integrated approach offers unique, in-depth insight into apoptotic dynamics that may prove important to preclinical evaluation of any anti-cancer compound.
Reciprocal virulence and resistance polymorphism in the relationship between Toxoplasma gondii and the house mouse [eLife recent issues] 2013-10-29 10:54
Virulence in the ubiquitous intracellular protozoon Toxoplasma gondii for its natural intermediate host, the mouse, appears paradoxical from an evolutionary standpoint because death of the mouse before encystment interrupts the parasite life cycle. Virulent T. gondii strains secrete kinases and pseudokinases that inactivate the immunity-related GTPases (IRG proteins) responsible for mouse resistance to avirulent strains. Such considerations stimulated a search for IRG alleles unknown in laboratory mice that might confer resistance to virulent strains of T. gondii. We report that the mouse IRG system shows extraordinary polymorphic complexity in the wild. We describe an IRG haplotype from a wild-derived mouse strain that confers resistance against virulent parasites by interference with the virulent kinase complex. In such hosts virulent strains can encyst, hinting at an explanation for the evolution of virulence polymorphism in T. gondii.
Structure and function of the Smoothened extracellular domain in vertebrate Hedgehog signaling [eLife recent issues] 2013-10-29 10:54
The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants.
Proteins pinpoint double strand breaks [eLife recent issues] 2013-10-29 10:54
Combining green fluorescent protein with a protein that only binds to double strand breaks in DNA allows these breaks—which are an important form of DNA damage—to be detected with high efficiency in living bacteria.
Immune to defeat [eLife recent issues] 2013-10-29 10:54
A dramatic example of the ‘arms race’ between hosts and pathogens has been observed in the response of mice to the parasite that causes toxoplasmosis.
Growth hormone-releasing hormone disruption extends lifespan and regulates response to caloric restriction in mice [eLife recent issues] 2013-10-29 10:54
We examine the impact of targeted disruption of growth hormone-releasing hormone (GHRH) in mice on longevity and the putative mechanisms of delayed aging. GHRH knockout mice are remarkably long-lived, exhibiting major shifts in the expression of genes related to xenobiotic detoxification, stress resistance, and insulin signaling. These mutant mice also have increased adiponectin levels and alterations in glucose homeostasis consistent with the removal of the counter-insulin effects of growth hormone. While these effects overlap with those of caloric restriction, we show that the effects of caloric restriction (CR) and the GHRH mutation are additive, with lifespan of GHRH-KO mutants further increased by CR. We conclude that GHRH-KO mice feature perturbations in a network of signaling pathways related to stress resistance, metabolic control and inflammation, and therefore provide a new model that can be used to explore links between GHRH repression, downregulation of the somatotropic axis, and extended longevity.
A genome-to-genome analysis of associations between human genetic variation, HIV-1 sequence diversity, and viral control [eLife recent issues] 2013-10-29 10:54
HIV-1 sequence diversity is affected by selection pressures arising from host genomic factors. Using paired human and viral data from 1071 individuals, we ran >3000 genome-wide scans, testing for associations between host DNA polymorphisms, HIV-1 sequence variation and plasma viral load (VL), while considering human and viral population structure. We observed significant human SNP associations to a total of 48 HIV-1 amino acid variants (p<2.4 x 10–12). All associated SNPs mapped to the HLA class I region. Clinical relevance of host and pathogen variation was assessed using VL results. We identified two critical advantages to the use of viral variation for identifying host factors: (1) association signals are much stronger for HIV-1 sequence variants than VL, reflecting the ‘intermediate phenotype’ nature of viral variation; (2) association testing can be run without any clinical data. The proposed genome-to-genome approach highlights sites of genomic conflict and is a strategy generally applicable to studies of host–pathogen interaction.
Conceptual metaphorical mapping in chimpanzees (Pan troglodytes) [eLife recent issues] 2013-10-22 14:54
Conceptual metaphors are linguistic constructions. Such a metaphor is humans’ mental representation of social rank as a pyramidal-like structure. High-ranked individuals are represented in higher positions than low-ranked individuals. We show that conceptual metaphorical mapping between social rank and the representational domain exists in our closest evolutionary relatives, the chimpanzees. Chimpanzee participants were requested to discriminate face identities in a vertical arrangement. We found a modulation of response latencies by the rank of the presented individual and the position on the display: a high-ranked individual presented in the higher and a low-ranked individual in the lower position led to quicker identity discrimination than a high-ranked individual in the lower and a low-ranked individual in the higher position. Such a spatial representation of dominance hierarchy in chimpanzees suggests that a natural tendency to systematically map an abstract dimension exists in the common ancestor of humans and chimpanzees.
Stochastic homeostasis in human airway epithelium is achieved by neutral competition of basal cell progenitors [eLife recent issues] 2013-10-22 14:54
Lineage tracing approaches have provided new insights into the cellular mechanisms that support tissue homeostasis in mice. However, the relevance of these discoveries to human epithelial homeostasis and its alterations in disease is unknown. By developing a novel quantitative approach for the analysis of somatic mitochondrial mutations that are accumulated over time, we demonstrate that the human upper airway epithelium is maintained by an equipotent basal progenitor cell population, in which the chance loss of cells due to lineage commitment is perfectly compensated by the duplication of neighbours, leading to "neutral drift" of the clone population. Further, we show that this process is accelerated in the airways of smokers, leading to intensified clonal consolidation and providing a background for tumorigenesis. This study provides a benchmark to show how somatic mutations provide quantitative information on homeostatic growth in human tissues, and a platform to explore factors leading to dysregulation and disease.
Multiple interfaces between a serine recombinase and an enhancer control site-specific DNA inversion [eLife recent issues] 2013-10-22 14:54
Serine recombinases are often tightly controlled by elaborate, topologically-defined, nucleoprotein complexes. Hin is a member of the DNA invertase subclass of serine recombinases that are regulated by a remote recombinational enhancer element containing two binding sites for the protein Fis. Two Hin dimers bound to specific recombination sites associate with the Fis-bound enhancer by DNA looping where they are remodeled into a synaptic tetramer competent for DNA chemistry and exchange. Here we show that the flexible beta-hairpin arms of the Fis dimers contact the DNA binding domain of one subunit of each Hin dimer. These contacts sandwich the Hin dimers to promote remodeling into the tetramer. A basic region on the Hin catalytic domain then contacts enhancer DNA to complete assembly of the active Hin tetramer. Our results reveal how the enhancer generates the recombination complex that specifies DNA inversion and regulates DNA exchange by the subunit rotation mechanism.
What silent mutations say about the human airways [eLife recent issues] 2013-10-22 14:54
A technique for tracing stem cells and their descendants reveals how the lining of the airways is maintained, and how this process is altered in smokers.
eLife and early career researchers [eLife recent issues] 2013-10-22 14:54
There are many reasons for submitting your best work to eLife, especially if you are an early career researcher.
Correction: A family of fluoride-specific ion channels with dual-topology architecture [eLife recent issues] 2013-10-22 14:54
A component of the mir-17-92 polycistronic oncomir promotes oncogene-dependent apoptosis [eLife recent issues] 2013-10-15 08:04
mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk.
Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling [eLife recent issues] 2013-10-15 08:04
Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped–Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation.
A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system [eLife recent issues] 2013-10-15 08:04
Establishing the time since death is critical in every death investigation, yet existing techniques are susceptible to a range of errors and biases. For example, forensic entomology is widely used to assess the postmortem interval (PMI), but errors can range from days to months. Microbes may provide a novel method for estimating PMI that avoids many of these limitations. Here we show that postmortem microbial community changes are dramatic, measurable, and repeatable in a mouse model system, allowing PMI to be estimated within approximately 3 days over 48 days. Our results provide a detailed understanding of bacterial and microbial eukaryotic ecology within a decomposing corpse system and suggest that microbial community data can be developed into a forensic tool for estimating PMI.
When 19 is greater than 92 [eLife recent issues] 2013-10-15 08:04
The gene miR-17-92 encodes six different microRNAs, with one of these acting as an internal brake that opposes the oncogenic activity of the others in some cancer contexts.
A year in the life of eLife [eLife recent issues] 2013-10-15 08:04
Improving the peer review process, overcoming the limitations of print journals and providing open access to the very best work in the life and biomedical sciences are three highlights of our first year.
Sense of achievement [eLife recent issues] 2013-10-15 08:04
Computational techniques developed to predict if odorants will interact with receptors in the olfactory system have achieved a success rate of 70%.
Correction: Nanoscale protein architecture of the kidney glomerular basement membrane [eLife recent issues] 2013-10-11 04:16
A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation [eLife recent issues] 2013-10-08 08:04
In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2–7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions.
A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity [eLife recent issues] 2013-10-08 08:04
RNA interference defends against viral infection in plant and animal cells. The nematode Caenorhabditis elegans and its natural pathogen, the positive-strand RNA virus Orsay, have recently emerged as a new animal model of host-virus interaction. Using a genome-wide association study in C. elegans wild populations and quantitative trait locus mapping, we identify a 159 base-pair deletion in the conserved drh-1 gene (encoding a RIG-I-like helicase) as a major determinant of viral sensitivity. We show that DRH-1 is required for the initiation of an antiviral RNAi pathway and the generation of virus-derived siRNAs (viRNAs). In mammals, RIG-I-domain containing proteins trigger an interferon-based innate immunity pathway in response to RNA virus infection. Our work in C. elegans demonstrates that the RIG-I domain has an ancient role in viral recognition. We propose that RIG-I acts as modular viral recognition factor that couples viral recognition to different effector pathways including RNAi and interferon responses.
Slo1 is the principal potassium channel of human spermatozoa [eLife recent issues] 2013-10-08 08:04
Mammalian spermatozoa gain competence to fertilize an oocyte as they travel through the female reproductive tract. This process is accompanied by an elevation of sperm intracellular calcium and a membrane hyperpolarization. The latter is evoked by K+ efflux; however, the molecular identity of the potassium channel of human spermatozoa (hKSper) is unknown. Here, we characterize hKSper, reporting that it is regulated by intracellular calcium but is insensitive to intracellular alkalinization. We also show that human KSper is inhibited by charybdotoxin, iberiotoxin, and paxilline, while mouse KSper is insensitive to these compounds. Such unique properties suggest that the Slo1 ion channel is the molecular determinant for hKSper. We show that Slo1 is localized to the sperm flagellum and is inhibited by progesterone. Inhibition of hKSper by progesterone may depolarize the spermatozoon to open the calcium channel CatSper, thus raising [Ca2+] to produce hyperactivation and allowing sperm to fertilize an oocyte.
Nanoscale protein architecture of the kidney glomerular basement membrane [eLife recent issues] 2013-10-08 08:04
In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM.
Sperm BerserKers [eLife recent issues] 2013-10-08 08:04
Human sperm cells rely on an unusual type of potassium ion channel.
Checkpoint proteins come under scrutiny [eLife recent issues] 2013-10-08 08:04
Details are emerging of the interactions between the kinetochore and various spindle checkpoint proteins that ensure that sister chromatids are equally divided between daughter cells during cell division.
Correction: Learning about loss [eLife recent issues] 2013-10-08 08:04
Cytoplasmic genetic variation and extensive cytonuclear interactions influence natural variation in the metabolome [eLife recent issues] 2013-10-08 08:04
Understanding genome to phenotype linkages has been greatly enabled by genomic sequencing. However, most genome analysis is typically confined to the nuclear genome. We conducted a metabolomic QTL analysis on a reciprocal RIL population structured to examine how variation in the organelle genomes affects phenotypic variation. This showed that the cytoplasmic variation had effects similar to, if not larger than, the largest individual nuclear locus. Inclusion of cytoplasmic variation into the genetic model greatly increased the explained phenotypic variation. Cytoplasmic genetic variation was a central hub in the epistatic network controlling the plant metabolome. This epistatic influence manifested such that the cytoplasmic background could alter or hide pairwise epistasis between nuclear loci. Thus, cytoplasmic genetic variation plays a central role in controlling natural variation in metabolomic networks. This suggests that cytoplasmic genomes must be included in any future analysis of natural variation.
ATR/Mec1 prevents lethal meiotic recombination initiation on partially replicated chromosomes in budding yeast [eLife recent issues] 2013-10-01 08:05
During gamete formation, crossover recombination must occur on replicated DNA to ensure proper chromosome segregation in the first meiotic division. We identified a Mec1/ATR- and Dbf4-dependent replication checkpoint in budding yeast that prevents the earliest stage of recombination, the programmed induction of DNA double-strand breaks (DSBs), when pre-meiotic DNA replication was delayed. The checkpoint acts through three complementary mechanisms: inhibition of Mer2 phosphorylation by Dbf4-dependent Cdc7 kinase, preclusion of chromosomal loading of Rec114 and Mre11, and lowered abundance of the Spo11 nuclease. Without this checkpoint, cells formed DSBs on partially replicated chromosomes. Importantly, such DSBs frequently failed to be repaired and impeded further DNA synthesis, leading to a rapid loss in cell viability. We conclude that a checkpoint-dependent constraint of DSB formation to duplicated DNA is critical not only for meiotic chromosome assortment, but also to protect genome integrity during gametogenesis.
The human gut and groundwater harbor non-photosynthetic bacteria belonging to a new candidate phylum sibling to Cyanobacteria [eLife recent issues] 2013-10-01 08:05
Cyanobacteria were responsible for the oxygenation of the ancient atmosphere; however, the evolution of this phylum is enigmatic, as relatives have not been characterized. Here we use whole genome reconstruction of human fecal and subsurface aquifer metagenomic samples to obtain complete genomes for members of a new candidate phylum sibling to Cyanobacteria, for which we propose the designation ‘Melainabacteria’. Metabolic analysis suggests that the ancestors to both lineages were non-photosynthetic, anaerobic, motile, and obligately fermentative. Cyanobacterial light sensing may have been facilitated by regulators present in the ancestor of these lineages. The subsurface organism has the capacity for nitrogen fixation using a nitrogenase distinct from that in Cyanobacteria, suggesting nitrogen fixation evolved separately in the two lineages. We hypothesize that Cyanobacteria split from Melainabacteria prior or due to the acquisition of oxygenic photosynthesis. Melainabacteria remained in anoxic zones and differentiated by niche adaptation, including for symbiosis in the mammalian gut.
Expanding the olfactory code by in silico decoding of odor-receptor chemical space [eLife recent issues] 2013-10-01 08:05
Coding of information in the peripheral olfactory system depends on two fundamental factors: interaction of individual odors with subsets of the odorant receptor repertoire and mode of signaling that an individual receptor-odor interaction elicits, activation or inhibition. We develop a cheminformatics pipeline that predicts receptor–odorant interactions from a large collection of chemical structures (>240,000) for receptors that have been tested to a smaller panel of odorants (~100). Using a computational approach, we first identify shared structural features from known ligands of individual receptors. We then use these features to screen in silico new candidate ligands from >240,000 potential volatiles for several Odorant receptors (Ors) in the Drosophila antenna. Functional experiments from 9 Ors support a high success rate (~71%) for the screen, resulting in identification of numerous new activators and inhibitors. Such computational prediction of receptor–odor interactions has the potential to enable systems level analysis of olfactory receptor repertoires in organisms.
A fair deal for PhD students and postdocs [eLife recent issues] 2013-10-01 08:05
The relentless expansion that threatens the sustainability of biomedical research in the US takes a heavy toll on young researchers.
Structural basis for activation and non-canonical catalysis of the Rap GTPase activating protein domain of plexin [eLife recent issues] 2013-10-01 08:05
Plexins are cell surface receptors that bind semaphorins and transduce signals for regulating neuronal axon guidance and other processes. Plexin signaling depends on their cytoplasmic GTPase activating protein (GAP) domain, which specifically inactivates the Ras homolog Rap through an ill-defined non-canonical catalytic mechanism. The plexin GAP is activated by semaphorin-induced dimerization, the structural basis for which remained unknown. Here we present the crystal structures of the active dimer of zebrafish PlexinC1 cytoplasmic region in the apo state and in complex with Rap. The structures show that the dimerization induces a large-scale conformational change in plexin, which opens the GAP active site to allow Rap binding. Plexin stabilizes the switch II region of Rap in an unprecedented conformation, bringing Gln63 in Rap into the active site for catalyzing GTP hydrolysis. The structures also explain the unique Rap-specificity of plexins. Mutational analyses support that these mechanisms underlie plexin activation and signaling.
Inter-Golgi transport mediated by COPI-containing vesicles carrying small cargoes [eLife recent issues] 2013-10-01 08:05
A core prediction of the vesicular transport model is that COPI vesicles are responsible for trafficking anterograde cargoes forward. In this study, we test this prediction by examining the properties and requirements of inter-Golgi transport within fused cells, which requires mobile carriers in order for exchange of constituents to occur. We report that both small soluble and membrane-bound secretory cargo and exogenous Golgi resident glycosyl-transferases are exchanged between separated Golgi. Large soluble aggregates, which traverse individual stacks, do not transfer between Golgi, implying that small cargoes (which can fit in a typical transport vesicle) are transported by a different mechanism. Super-resolution microscopy reveals that the carriers of both anterograde and retrograde cargoes are the size of COPI vesicles, contain coatomer, and functionally require ARF1 and coatomer for transport. The data suggest that COPI vesicles traffic both small secretory cargo and steady-state Golgi resident enzymes among stacked cisternae that are stationary.
Phenotypic landscape inference reveals multiple evolutionary paths to C4 photosynthesis [eLife recent issues] 2013-09-28 04:33
C4 photosynthesis has independently evolved from the ancestral C3 pathway in at least 60 plant lineages, but, as with other complex traits, how it evolved is unclear. Here we show that the polyphyletic appearance of C4 photosynthesis is associated with diverse and flexible evolutionary paths that group into four major trajectories. We conducted a meta-analysis of 18 lineages containing species that use C3, C4, or intermediate C3–C4 forms of photosynthesis to parameterise a 16-dimensional phenotypic landscape. We then developed and experimentally verified a novel Bayesian approach based on a hidden Markov model that predicts how the C4 phenotype evolved. The alternative evolutionary histories underlying the appearance of C4 photosynthesis were determined by ancestral lineage and initial phenotypic alterations unrelated to photosynthesis. We conclude that the order of C4 trait acquisition is flexible and driven by non-photosynthetic drivers. This flexibility will have facilitated the convergent evolution of this complex trait.
Shining fresh light on the evolution of photosynthesis [eLife recent issues] 2013-09-28 04:33
There are two types of photosynthesis, C3 and C4, and computational techniques have been used to explore how C4 plants evolved from their C3 ancestors.
Direct observation of frequency modulated transcription in single cells using light activation [eLife recent issues] 2013-09-24 11:44
Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.
Cavin-3 dictates the balance between ERK and Akt signaling [eLife recent issues] 2013-09-24 11:44
Cavin-3 is a tumor suppressor protein of unknown function. Using both in vivo and in vitro approaches, we show that cavin-3 dictates the balance between ERK and Akt signaling. Loss of cavin-3 increases Akt signaling at the expense of ERK, while gain of cavin-3 increases ERK signaling at the expense Akt. Cavin-3 facilitates signal transduction to ERK by anchoring caveolae to the membrane skeleton of the plasma membrane via myosin-1c. Caveolae are lipid raft specializations that contain an ERK activation module and loss of the cavin-3 linkage reduces the abundance of caveolae, thereby separating this ERK activation module from signaling receptors. Loss of cavin-3 promotes Akt signaling through suppression of EGR1 and PTEN. The in vitro consequences of the loss of cavin-3 include induction of Warburg metabolism (aerobic glycolysis), accelerated cell proliferation, and resistance to apoptosis. The in vivo consequences of cavin-3 knockout are increased lactate production and cachexia.
Complete dissection of transcription elongation reveals slow translocation of RNA polymerase II in a linear ratchet mechanism [eLife recent issues] 2013-09-24 11:44
During transcription elongation, RNA polymerase has been assumed to attain equilibrium between pre- and post-translocated states rapidly relative to the subsequent catalysis. Under this assumption, recent single-molecule studies proposed a branched Brownian ratchet mechanism that necessitates a putative secondary nucleotide binding site on the enzyme. By challenging individual yeast RNA polymerase II with a nucleosomal barrier, we separately measured the forward and reverse translocation rates. Surprisingly, we found that the forward translocation rate is comparable to the catalysis rate. This finding reveals a linear, non-branched ratchet mechanism for the nucleotide addition cycle in which translocation is one of the rate-limiting steps. We further determined all the major on- and off-pathway kinetic parameters in the elongation cycle. The resulting translocation energy landscape shows that the off-pathway states are favored thermodynamically but not kinetically over the on-pathway states, conferring the enzyme its propensity to pause and furnishing the physical basis for transcriptional regulation.
Apoptotic cells can induce non-autonomous apoptosis through the TNF pathway [eLife recent issues] 2013-09-24 11:44
Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. We identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. We identified Eiger, the Drosophila tumor necrosis factor (TNF) homolog, as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. We also show that during coordinated cell death of hair follicle cells in mice, TNF-α is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions.
Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling [eLife recent issues] 2013-09-24 11:44
Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores.
Eiger triggers death from afar [eLife recent issues] 2013-09-24 11:44
Cells undergoing programmed cell death release signals that can trigger the death of cells at remote locations.
On the move [eLife recent issues] 2013-09-24 11:44
Single-molecule experiments have shed new light on the mechanisms responsible for the movement of RNA polymerase along DNA during transcription.
A new regulator of caveolae signalling [eLife recent issues] 2013-09-24 11:44
Cavin-3 regulates metabolism and cell proliferation by coordinating the activities of growth factor signalling cascades.
Caveolae internalization repairs wounded cells and muscle fibers [eLife recent issues] 2013-09-17 08:03
Rapid repair of plasma membrane wounds is critical for cellular survival. Muscle fibers are particularly susceptible to injury, and defective sarcolemma resealing causes muscular dystrophy. Caveolae accumulate in dystrophic muscle fibers and caveolin and cavin mutations cause muscle pathology, but the underlying mechanism is unknown. Here we show that muscle fibers and other cell types repair membrane wounds by a mechanism involving Ca2+-triggered exocytosis of lysosomes, release of acid sphingomyelinase, and rapid lesion removal by caveolar endocytosis. Wounding or exposure to sphingomyelinase triggered endocytosis and intracellular accumulation of caveolar vesicles, which gradually merged into larger compartments. The pore-forming toxin SLO was directly visualized entering cells within caveolar vesicles, and depletion of caveolin inhibited plasma membrane resealing. Our findings directly link lesion removal by caveolar endocytosis to the maintenance of plasma membrane and muscle fiber integrity, providing a mechanistic explanation for the muscle pathology associated with mutations in caveolae proteins.
The structure of the COPII transport-vesicle coat assembled on membranes [eLife recent issues] 2013-09-17 08:03
Coat protein complex II (COPII) mediates formation of the membrane vesicles that export newly synthesised proteins from the endoplasmic reticulum. The inner COPII proteins bind to cargo and membrane, linking them to the outer COPII components that form a cage around the vesicle. Regulated flexibility in coat architecture is essential for transport of a variety of differently sized cargoes, but structural data on the assembled coat has not been available. We have used cryo-electron tomography and subtomogram averaging to determine the structure of the complete, membrane-assembled COPII coat. We describe a novel arrangement of the outer coat and find that the inner coat can assemble into regular lattices. The data reveal how coat subunits interact with one another and with the membrane, suggesting how coordinated assembly of inner and outer coats can mediate and regulate packaging of vesicles ranging from small spheres to large tubular carriers.
Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly [eLife recent issues] 2013-09-17 08:03
Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis.
The antigenic switching network of Plasmodium falciparum and its implications for the immuno-epidemiology of malaria [eLife recent issues] 2013-09-17 08:03
Antigenic variation in the human malaria parasite Plasmodium falciparum involves sequential and mutually exclusive expression of members of the var multi-gene family and appears to follow a non-random pattern. In this study, using a detailed in vitro gene transcription analysis of the culture-adapted HB3 strain of P. falciparum, we show that antigenic switching is governed by a global activation hierarchy favouring short and highly diverse genes in central chromosomal location. Longer and more conserved genes, which have previously been associated with severe infection in immunologically naive hosts, are rarely activated, however, implying an in vivo fitness advantage possibly through adhesion-dependent survival rates. We further show that a gene’s activation rate is positively associated sequence diversity, which could offer important new insights into the evolution and maintenance of antigenic diversity in P. falciparum malaria.
MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons [eLife recent issues] 2013-09-17 08:03
Bone morphogenetic protein (BMP) signaling has emerged as an important regulator of sensory neuron development. Using a three-generation forward genetic screen in mice we have identified Megf8 as a novel modifier of BMP4 signaling in trigeminal ganglion (TG) neurons. Loss of Megf8 disrupts axon guidance in the peripheral nervous system and leads to defects in development of the limb, heart, and left-right patterning, defects that resemble those observed in Bmp4 loss-of-function mice. Bmp4 is expressed in a pattern that defines the permissive field for the peripheral projections of TG axons and mice lacking BMP signaling in sensory neurons exhibit TG axon defects that resemble those observed in Megf8–/– embryos. Furthermore, TG axon growth is robustly inhibited by BMP4 and this inhibition is dependent on Megf8. Thus, our data suggest that Megf8 is involved in mediating BMP4 signaling and guidance of developing TG axons.
Tissue absence initiates regeneration through Follistatin-mediated inhibition of Activin signaling [eLife recent issues] 2013-09-10 08:03
Regeneration is widespread, but mechanisms that activate regeneration remain mysterious. Planarians are capable of whole-body regeneration and mount distinct molecular responses to wounds that result in tissue absence and those that do not. A major question is how these distinct responses are activated. We describe a follistatin homolog (Smed-follistatin) required for planarian regeneration. Smed-follistatin inhibition blocks responses to tissue absence but does not prevent normal tissue turnover. Two activin homologs (Smed-activin-1 and Smed-activin-2) are required for the Smed-follistatin phenotype. Finally, Smed-follistatin is wound-induced and expressed at higher levels following injuries that cause tissue absence. These data suggest that Smed-follistatin inhibits Smed-Activin proteins to trigger regeneration specifically following injuries involving tissue absence and identify a mechanism critical for regeneration initiation, a process important across the animal kingdom.
Imaging-based chemical screening reveals activity-dependent neural differentiation of pluripotent stem cells [eLife recent issues] 2013-09-10 08:03
Mammalian pluripotent stem cells (PSCs) represent an important venue for understanding basic principles regulating tissue-specific differentiation and discovering new tools that may facilitate clinical applications. Mechanisms that direct neural differentiation of PSCs involve growth factor signaling and transcription regulation. However, it is unknown whether and how electrical activity influences this process. Here we report a high throughput imaging-based screen, which uncovers that selamectin, an anti-helminthic therapeutic compound with reported activity on invertebrate glutamate-gated chloride channels, promotes neural differentiation of PSCs. We show that selamectin’s pro-neurogenic activity is mediated by 2-containing GABAA receptors in subsets of neural rosette progenitors, accompanied by increased proneural and lineage-specific transcription factor expression and cell cycle exit. In vivo, selamectin promotes neurogenesis in developing zebrafish. Our results establish a chemical screening platform that reveals activity-dependent neural differentiation from PSCs. Compounds identified in this and future screening might prove therapeutically beneficial for treating neurodevelopmental or neurodegenerative disorders.
Learning about loss [eLife recent issues] 2013-09-10 08:03
Researchers have identified two genes—follistatin and activin—as having an important role in the ability of certain flatworms to identify wounds that require the production of new tissue.
An essential role for maternal control of Nodal signaling [eLife recent issues] 2013-09-10 08:03
Growth factor signaling is essential for pattern formation, growth, differentiation, and maintenance of stem cell pluripotency. Nodal-related signaling factors are required for axis formation and germ layer specification from sea urchins to mammals. Maternal transcripts of the zebrafish Nodal factor, Squint (Sqt), are localized to future embryonic dorsal. The mechanisms by which maternal sqt/nodal RNA is localized and regulated have been unclear. Here, we show that maternal control of Nodal signaling via the conserved Y box-binding protein 1 (Ybx1) is essential. We identified Ybx1 via a proteomic screen. Ybx1 recognizes the 3’ untranslated region (UTR) of sqt RNA and prevents premature translation and Sqt/Nodal signaling. Maternal-effect mutations in zebrafish ybx1 lead to deregulated Nodal signaling, gastrulation failure, and embryonic lethality. Implanted Nodal-coated beads phenocopy ybx1 mutant defects. Thus, Ybx1 prevents ectopic Nodal activity, revealing a new paradigm in the regulation of Nodal signaling, which is likely to be conserved.
Collaboration gets the most out of software [eLife recent issues] 2013-09-10 08:03
By centralizing many of the tasks associated with the upkeep of scientific software, SBGrid allows researchers to spend more of their time on research.
Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species [eLife recent issues] 2013-09-03 16:45
Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range.
Ultrafast endocytosis at Caenorhabditis elegans neuromuscular junctions [eLife recent issues] 2013-09-03 16:45
Synaptic vesicles can be released at extremely high rates, which places an extraordinary demand on the recycling machinery. Previous ultrastructural studies of vesicle recycling were conducted in dissected preparations using an intense stimulation to maximize the probability of release. Here, a single light stimulus was applied to motor neurons in intact Caenorhabditis elegans nematodes expressing channelrhodopsin, and the animals rapidly frozen. We found that docked vesicles fuse along a broad active zone in response to a single stimulus, and are replenished with a time constant of about 2 s. Endocytosis occurs within 50 ms adjacent to the dense projection and after 1 s adjacent to adherens junctions. These studies suggest that synaptic vesicle endocytosis may occur on a millisecond time scale following a single physiological stimulus in the intact nervous system and is unlikely to conform to current models of endocytosis.
DNA methylation presents distinct binding sites for human transcription factors [eLife recent issues] 2013-09-03 16:45
DNA methylation, especially CpG methylation at promoter regions, has been generally considered as a potent epigenetic modification that prohibits transcription factor (TF) recruitment, resulting in transcription suppression. Here, we used a protein microarray-based approach to systematically survey the entire human TF family and found numerous purified TFs with methylated CpG (mCpG)-dependent DNA-binding activities. Interestingly, some TFs exhibit specific binding activity to methylated and unmethylated DNA motifs of distinct sequences. To elucidate the underlying mechanism, we focused on Kruppel-like factor 4 (KLF4), and decoupled its mCpG- and CpG-binding activities via site-directed mutagenesis. Furthermore, KLF4 binds specific methylated or unmethylated motifs in human embryonic stem cells in vivo. Our study suggests that mCpG-dependent TF binding activity is a widespread phenomenon and provides a new framework to understand the role and mechanism of TFs in epigenetic regulation of gene transcription.
High-order social interactions in groups of mice [eLife recent issues] 2013-09-03 16:45
Social behavior in mammals is often studied in pairs under artificial conditions, yet groups may rely on more complicated social structures. Here, we use a novel system for tracking multiple animals in a rich environment to characterize the nature of group behavior and interactions, and show strongly correlated group behavior in mice. We have found that the minimal models that rely only on individual traits and pairwise correlations between animals are not enough to capture group behavior, but that models that include third-order interactions give a very accurate description of the group. These models allow us to infer social interaction maps for individual groups. Using this approach, we show that environmental complexity during adolescence affects the collective group behavior of adult mice, in particular altering the role of high-order structure. Our results provide new experimental and mathematical frameworks for studying group behavior and social interactions.
Mechanism of synergistic activation of Arp2/3 complex by cortactin and N-WASP [eLife recent issues] 2013-09-03 16:45
Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a ~160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation.
Cytoplasmic dynein crosslinks and slides anti-parallel microtubules using its two motor domains [eLife recent issues] 2013-09-03 16:45
Cytoplasmic dynein is the predominant minus-end-directed microtubule (MT) motor in most eukaryotic cells. In addition to transporting vesicular cargos, dynein helps to organize MTs within MT networks such as mitotic spindles. How dynein performs such non-canonical functions is unknown. Here we demonstrate that dynein crosslinks and slides anti-parallel MTs in vitro. Surprisingly, a minimal dimeric motor lacking a tail domain and associated subunits can cause MT sliding. Single molecule imaging reveals that motors pause and frequently reverse direction when encountering an anti-parallel MT overlap, suggesting that the two motor domains can bind both MTs simultaneously. In the mitotic spindle, inward microtubule sliding by dynein counteracts outward sliding generated by kinesin-5, and we show that a tailless, dimeric motor is sufficient to drive this activity in mammalian cells. Our results identify an unexpected mechanism for dynein-driven microtubule sliding, which differs from filament sliding mechanisms described for other motor proteins.
Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation [eLife recent issues] 2013-09-03 16:45
During cell locomotion and endocytosis, membrane-tethered WASP proteins stimulate actin filament nucleation by the Arp2/3 complex. This process generates highly branched arrays of filaments that grow toward the membrane to which they are tethered, a conflict that seemingly would restrict filament growth. Using three-color single-molecule imaging in vitro we revealed how the dynamic associations of Arp2/3 complex with mother filament and WASP are temporally coordinated with initiation of daughter filament growth. We found that WASP proteins dissociated from filament-bound Arp2/3 complex prior to new filament growth. Further, mutations that accelerated release of WASP from filament-bound Arp2/3 complex proportionally accelerated branch formation. These data suggest that while WASP promotes formation of pre-nucleation complexes, filament growth cannot occur until it is triggered by WASP release. This provides a mechanism by which membrane-bound WASP proteins can stimulate network growth without restraining it.
The tortoise and the hare revisited [eLife recent issues] 2013-09-03 16:45
Optogenetics and electron microscopy reveal an ultrafast mode of synaptic vesicle recycling, adding a new twist to a 40-year-old controversy.
Making the most of methylation [eLife recent issues] 2013-09-03 16:45
A high-throughput screening approach based on a protein microarray reveals that many human transcription factors interact specifically with methylated promoter sequences.
Correction: A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network [eLife recent issues] 2013-08-28 12:24
A family of fluoride-specific ion channels with dual-topology architecture [eLife recent issues] 2013-08-27 10:55
Fluoride ion, ubiquitous in soil, water, and marine environments, is a chronic threat to microorganisms. Many prokaryotes, archea, unicellular eukaryotes, and plants use a recently discovered family of F– exporter proteins to lower cytoplasmic F– levels to counteract the anion’s toxicity. We show here that these ‘Fluc’ proteins, purified and reconstituted in liposomes and planar phospholipid bilayers, form constitutively open anion channels with extreme selectivity for F– over Cl–. The active channel is a dimer of identical or homologous subunits arranged in antiparallel transmembrane orientation. This dual-topology assembly has not previously been seen in ion channels but is known in multidrug transporters of the SMR family, and is suggestive of an evolutionary antecedent of the inverted repeats found within the subunits of many membrane transport proteins.
Stem cells set their sights on retinitis pigmentosa [eLife recent issues] 2013-08-27 10:55
Skin cells from a patient with a form of inherited blindness have been reprogrammed into retinal cells and successfully transplanted into mice.
Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice [eLife recent issues] 2013-08-27 10:55
A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.
Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa [eLife recent issues] 2013-08-27 10:55
Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1–/– mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.
Physiological and stem cell compartmentalization within the Drosophila midgut [eLife recent issues] 2013-08-27 10:55
The Drosophila midgut is maintained throughout its length by superficially similar, multipotent intestinal stem cells that generate new enterocytes and enteroendocrine cells in response to tissue requirements. We found that the midgut shows striking regional differentiation along its anterior-posterior axis. At least ten distinct subregions differ in cell morphology, physiology and the expression of hundreds of genes with likely tissue functions. Stem cells also vary regionally in behavior and gene expression, suggesting that they contribute to midgut sub-specialization. Clonal analyses showed that stem cells generate progeny located outside their own subregion at only one of six borders tested, suggesting that midgut subregions resemble cellular compartments involved in tissue development. Tumors generated by disrupting Notch signaling arose preferentially in three subregions and tumor cells also appeared to respect regional borders. Thus, apparently similar intestinal stem cells differ regionally in cell production, gene expression and in the ability to spawn tumors.
Assembly, remodelled [eLife recent issues] 2013-08-20 11:07
Biochemical assays reveal that nucleosome maturation and chromatin remodelling by the motor protein Chd1 are distinct, separable enzymatic activities.
APP interacts with LRP4 and agrin to coordinate the development of the neuromuscular junction in mice [eLife recent issues] 2013-08-20 08:52
ApoE, ApoE receptors and APP cooperate in the pathogenesis of Alzheimer’s disease. Intriguingly, the ApoE receptor LRP4 and APP are also required for normal formation and function of the neuromuscular junction (NMJ). In this study, we show that APP interacts with LRP4, an obligate co-receptor for muscle-specific tyrosine kinase (MuSK). Agrin, a ligand for LRP4, also binds to APP and co-operatively enhances the interaction of APP with LRP4. In cultured myotubes, APP synergistically increases agrin-induced acetylcholine receptor (AChR) clustering. Deletion of the transmembrane domain of LRP4 (LRP4 ECD) results in growth retardation of the NMJ, and these defects are markedly enhanced in APP–/–;LRP4ECD/ECD mice. Double mutant NMJs are significantly reduced in size and number, resulting in perinatal lethality. Our findings reveal novel roles for APP in regulating neuromuscular synapse formation through hetero-oligomeric interaction with LRP4 and agrin and thereby provide new insights into the molecular mechanisms that govern NMJ formation and maintenance.
ATP-dependent chromatin assembly is functionally distinct from chromatin remodeling [eLife recent issues] 2013-08-20 08:52
Chromatin assembly involves the combined action of ATP-dependent motor proteins and histone chaperones. Because motor proteins in chromatin assembly also function as chromatin remodeling factors, we investigated the relationship between ATP-driven chromatin assembly and chromatin remodeling in the generation of periodic nucleosome arrays. We found that chromatin remodeling-defective Chd1 motor proteins are able to catalyze ATP-dependent chromatin assembly. The resulting nucleosomes are not, however, spaced in periodic arrays. Wild-type Chd1, but not chromatin remodeling-defective Chd1, can catalyze the conversion of randomly-distributed nucleosomes into periodic arrays. These results reveal a functional distinction between ATP-dependent nucleosome assembly and chromatin remodeling, and suggest a model for chromatin assembly in which randomly-distributed nucleosomes are formed by the nucleosome assembly function of Chd1, and then regularly-spaced nucleosome arrays are generated by the chromatin remodeling activity of Chd1. These findings uncover an unforeseen level of specificity in the role of motor proteins in chromatin assembly.
Endocytosis gets in tune with action potential bursts [eLife recent issues] 2013-08-20 08:52
Neurons use a calcium-dependent mechanism to optimize the rate at which synaptic vesicles are recycled.
MAVS recruits multiple ubiquitin E3 ligases to activate antiviral signaling cascades [eLife recent issues] 2013-08-14 09:34
RNA virus infections are detected by the RIG-I family of receptors, which induce type-I interferons through the mitochondrial protein MAVS. MAVS forms large prion-like polymers that activate the cytosolic kinases IKK and TBK1, which in turn activate NF-B and IRF3, respectively, to induce interferons. Here we show that MAVS polymers recruit several TRAF proteins, including TRAF2, TRAF5, and TRAF6, through distinct TRAF-binding motifs. Mutations of these motifs that disrupted MAVS binding to TRAFs abrogated its ability to activate IRF3. IRF3 activation was also abolished in cells lacking TRAF2, 5, and 6. These TRAF proteins promoted ubiquitination reactions that recruited NEMO to the MAVS signaling complex, leading to the activation of IKK and TBK1. These results delineate the mechanism of MAVS signaling and reveal that TRAF2, 5, and 6, which are normally associated with NF-B activation, also play a crucial role in IRF3 activation in antiviral immune responses.
A global change in RNA polymerase II pausing during the Drosophila midblastula transition [eLife recent issues] 2013-08-13 11:01
Massive zygotic transcription begins in many organisms during the midblastula transition when the cell cycle of the dividing egg slows down. A few genes are transcribed before this stage but how this differential activation is accomplished is still an open question. We have performed ChIP-seq experiments on tightly staged Drosophila embryos and show that massive recruitment of RNA polymerase II (Pol II) with widespread pausing occurs de novo during the midblastula transition. However, ~100 genes are strongly occupied by Pol II before this timepoint and most of them do not show Pol II pausing, consistent with a requirement for rapid transcription during the fast nuclear cycles. This global change in Pol II pausing correlates with distinct core promoter elements and associates a TATA-enriched promoter with the rapid early transcription. This suggests that promoters are differentially used during the zygotic genome activation, presumably because they have distinct dynamic properties.
UNC-13L, UNC-13S, and Tomosyn form a protein code for fast and slow neurotransmitter release in Caenorhabditis elegans [eLife recent issues] 2013-08-13 11:01
Synaptic transmission consists of fast and slow components of neurotransmitter release. Here we show that these components are mediated by distinct exocytic proteins. The Caenorhabditis elegans unc-13 gene is required for SV exocytosis, and encodes long and short isoforms (UNC-13L and S). Fast release was mediated by UNC-13L, whereas slow release required both UNC-13 proteins and was inhibited by Tomosyn. The spatial location of each protein correlated with its effect. Proteins adjacent to the dense projection mediated fast release, while those controlling slow release were more distal or diffuse. Two UNC-13L domains accelerated release. C2A, which binds RIM (a protein associated with calcium channels), anchored UNC-13 at active zones and shortened the latency of release. A calmodulin binding site accelerated release but had little effect on UNC-13’s spatial localization. These results suggest that UNC-13L, UNC-13S, and Tomosyn form a molecular code that dictates the timing of neurotransmitter release.
Changing the rules of the game [eLife recent issues] 2013-08-13 11:01
Genomics researchers have built a Facebook game that allows members of the public to join the effort to understand a disease that has killed millions of ash trees across Europe.
Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3 [eLife recent issues] 2013-08-08 09:02
Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2~ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT~ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3~ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3~ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation.
Caught in the act [eLife recent issues] 2013-08-08 09:02
The crystal structure of a HECT E3 enzyme has been captured as it transfers ubiquitin to a target protein, revealing the dramatic changes in shape that enable it to modify particular residues in its targets.
Cellular resolution models for even skipped regulation in the entire Drosophila embryo [eLife recent issues] 2013-08-06 09:57
Transcriptional control ensures genes are expressed in the right amounts at the correct times and locations. Understanding quantitatively how regulatory systems convert input signals to appropriate outputs remains a challenge. For the first time, we successfully model even skipped (eve) stripes 2 and 3+7 across the entire fly embryo at cellular resolution. A straightforward statistical relationship explains how transcription factor (TF) concentrations define eve’s complex spatial expression, without the need for pairwise interactions or cross-regulatory dynamics. Simulating thousands of TF combinations, we recover known regulators and suggest new candidates. Finally, we accurately predict the intricate effects of perturbations including TF mutations and misexpression. Our approach imposes minimal assumptions about regulatory function; instead we infer underlying mechanisms from models that best fit the data, like the lack of TF-specific thresholds and the positional value of homotypic interactions. Our study provides a general and quantitative method for elucidating the regulation of diverse biological systems.
Single molecule imaging reveals a major role for diffusion in the exploration of ciliary space by signaling receptors [eLife recent issues] 2013-08-06 09:57
The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined.
The ER-Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis [eLife recent issues] 2013-08-06 09:57
Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.
Computing away the magic? [eLife recent issues] 2013-08-06 09:57
Computer simulations and quantitative imaging of Drosophila embryos have been used to recreate the dynamic activities of a complex transcriptional enhancer.
Functional genomic characterization of neoblast-like stem cells in larval Schistosoma mansoni [eLife recent issues] 2013-07-30 11:03
Schistosomes infect hundreds of millions of people in the developing world. Transmission of these parasites relies on a stem cell-driven, clonal expansion of larvae inside a molluscan intermediate host. How this novel asexual reproductive strategy relates to current models of stem cell maintenance and germline specification is unclear. Here, we demonstrate that this proliferative larval cell population (germinal cells) shares some molecular signatures with stem cells from diverse organisms, in particular neoblasts of planarians (free-living relatives of schistosomes). We identify two distinct germinal cell lineages that differ in their proliferation kinetics and expression of a nanos ortholog. We show that a vasa/PL10 homolog is required for proliferation and maintenance of both populations, whereas argonaute2 and a fibroblast growth factor receptor-encoding gene are required only for nanos-negative cells. Our results suggest that an ancient stem cell-based developmental program may have enabled the evolution of the complex life cycle of parasitic flatworms.
Minor introns are embedded molecular switches regulated by highly unstable U6atac snRNA [eLife recent issues] 2013-07-30 11:03
Eukaryotes have two types of spliceosomes, comprised of either major (U1, U2, U4, U5, U6) or minor (U11, U12, U4atac, U6atac; <1%) snRNPs. The high conservation of minor introns, typically one amidst many major introns in several hundred genes, despite their poor splicing, has been a long-standing enigma. Here, we discovered that the low abundance minor spliceosome’s catalytic snRNP, U6atac, is strikingly unstable (t1/2<2 hr). We show that U6atac level depends on both RNA polymerases II and III and can be rapidly increased by cell stress-activated kinase p38MAPK, which stabilizes it, enhancing mRNA expression of hundreds of minor intron-containing genes that are otherwise suppressed by limiting U6atac. Furthermore, p38MAPK-dependent U6atac modulation can control minor intron-containing tumor suppressor PTEN expression and cytokine production. We propose that minor introns are embedded molecular switches regulated by U6atac abundance, providing a novel post-transcriptional gene expression mechanism and a rationale for the minor spliceosome’s evolutionary conservation.
In situ structural analysis of the Yersinia enterocolitica injectisome [eLife recent issues] 2013-07-30 11:03
Injectisomes are multi-protein transmembrane machines allowing pathogenic bacteria to inject effector proteins into eukaryotic host cells, a process called type III secretion. Here we present the first three-dimensional structure of Yersinia enterocolitica and Shigella flexneri injectisomes in situ and the first structural analysis of the Yersinia injectisome. Unexpectedly, basal bodies of injectisomes inside the bacterial cells showed length variations of 20%. The in situ structures of the Y. enterocolitica and S. flexneri injectisomes had similar dimensions and were significantly longer than the isolated structures of related injectisomes. The crystal structure of the inner membrane injectisome component YscD appeared elongated compared to a homologous protein, and molecular dynamics simulations documented its elongation elasticity. The ring-shaped secretin YscC at the outer membrane was stretched by 30–40% in situ, compared to its isolated liposome-embedded conformation. We suggest that elasticity is critical for some two-membrane spanning protein complexes to cope with variations in the intermembrane distance.
Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1 [eLife recent issues] 2013-07-30 11:03
RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals.
Dynamin phosphorylation controls optimization of endocytosis for brief action potential bursts [eLife recent issues] 2013-07-30 11:03
Modulation of synaptic vesicle retrieval is considered to be potentially important in steady-state synaptic performance. Here we show that at physiological temperature endocytosis kinetics at hippocampal and cortical nerve terminals show a bi-phasic dependence on electrical activity. Endocytosis accelerates for the first 15–25 APs during bursts of action potential firing, after which it slows with increasing burst length creating an optimum stimulus for this kinetic parameter. We show that activity-dependent acceleration is only prominent at physiological temperature and that the mechanism of this modulation is based on the dephosphorylation of dynamin 1. Nerve terminals in which dynamin 1 and 3 have been replaced with dynamin 1 harboring dephospho- or phospho-mimetic mutations in the proline-rich domain eliminate the acceleration phase by either setting endocytosis at an accelerated state or a decelerated state, respectively.
On the trail of a tropical disease [eLife recent issues] 2013-07-30 11:03
Learning more about the cells that enable parasitic worms called schistosomes to reproduce inside snails could lead to new treatments that prevent these parasites from being transmitted to humans.
How to switch a master switch [eLife recent issues] 2013-07-30 11:03
The crystal structure of a nucleotide exchange factor in white blood cells reveals an autoinhibitory mechanism that reinforces the switch-like behaviour of the signalling protein Ras.
Segregation of complex acoustic scenes based on temporal coherence [eLife recent issues] 2013-07-23 12:27
In contrast to the complex acoustic environments we encounter everyday, most studies of auditory segregation have used relatively simple signals. Here, we synthesized a new stimulus to examine the detection of coherent patterns (‘figures’) from overlapping ‘background’ signals. In a series of experiments, we demonstrate that human listeners are remarkably sensitive to the emergence of such figures and can tolerate a variety of spectral and temporal perturbations. This robust behavior is consistent with the existence of automatic auditory segregation mechanisms that are highly sensitive to correlations across frequency and time. The observed behavior cannot be explained purely on the basis of adaptation-based models used to explain the segregation of deterministic narrowband signals. We show that the present results are consistent with the predictions of a model of auditory perceptual organization based on temporal coherence. Our data thus support a role for temporal coherence as an organizational principle underlying auditory segregation.
A mammalian pseudogene lncRNA at the interface of inflammation and anti-inflammatory therapeutics [eLife recent issues] 2013-07-23 12:27
Pseudogenes are thought to be inactive gene sequences, but recent evidence of extensive pseudogene transcription raised the question of potential function. Here we discover and characterize the sets of mouse lncRNAs induced by inflammatory signaling via TNFα. TNFα regulates hundreds of lncRNAs, including 54 pseudogene lncRNAs, several of which show exquisitely selective expression in response to specific cytokines and microbial components in a NF-B-dependent manner. Lethe, a pseudogene lncRNA, is selectively induced by proinflammatory cytokines via NF-B or glucocorticoid receptor agonist, and functions in negative feedback signaling to NF-B. Lethe interacts with NF-B subunit RelA to inhibit RelA DNA binding and target gene activation. Lethe level decreases with organismal age, a physiological state associated with increased NF-B activity. These findings suggest that expression of pseudogenes lncRNAs are actively regulated and constitute functional regulators of inflammatory signaling.
Direct live imaging of cell-cell protein transfer by transient outer membrane fusion in Myxococcus xanthus [eLife recent issues] 2013-07-23 12:27
In bacteria, multicellular behaviors are regulated by cell–cell signaling through the exchange of both diffusible and contact-dependent signals. In a multicellular context, Myxococcus cells can share outer membrane (OM) materials by an unknown mechanism involving the traAB genes and gliding motility. Using live imaging, we show for the first time that transient contacts between two cells are sufficient to transfer OM materials, proteins and lipids, at high efficiency. Transfer was associated with the formation of dynamic OM tubes, strongly suggesting that transfer results from the local fusion of the OMs of two transferring cells. Last, large amounts of OM materials were released in slime trails deposited by gliding cells. Since cells tend to follow trails laid by other cells, slime-driven OM material exchange may be an important stigmergic regulation of Myxococcus social behaviors.
Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4 [eLife recent issues] 2013-07-23 12:27
Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway.
Time is of the essence for auditory scene analysis [eLife recent issues] 2013-07-23 12:27
Using computational models and stimuli that resemble natural acoustic signals, auditory scientists explore how we segregate competing streams of sound.
Grandmother elephants [eLife recent issues] 2013-07-23 12:27
As new technology makes it possible to perform experiments that were unimaginable a decade ago, Eve Marder argues that we can still learn from the past.
Correction: Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [eLife recent issues] 2013-07-23 12:27
Correction: Closing in on a new treatment for sleeping sickness [eLife recent issues] 2013-07-23 12:27
Correction: Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta [eLife recent issues] 2013-07-23 12:27
Integrative genomic analysis of the human immune response to influenza vaccination [eLife recent issues] 2013-07-16 12:17
Identification of the host genetic factors that contribute to variation in vaccine responsiveness may uncover important mechanisms affecting vaccine efficacy. We carried out an integrative, longitudinal study combining genetic, transcriptional, and immunologic data in humans given seasonal influenza vaccine. We identified 20 genes exhibiting a transcriptional response to vaccination, significant genotype effects on gene expression, and correlation between the transcriptional and antibody responses. The results show that variation at the level of genes involved in membrane trafficking and antigen processing significantly influences the human response to influenza vaccination. More broadly, we demonstrate that an integrative study design is an efficient alternative to existing methods for the identification of genes involved in complex traits.
The insulin receptor cellular IRES confers resistance to eIF4A inhibition [eLife recent issues] 2013-07-16 12:17
Under conditions of stress, such as limited growth factor signaling, translation is inhibited by the action of 4E-BP and PDCD4. These proteins, through inhibition of eIF4E and eIF4A, respectively, impair cap-dependent translation. Under stress conditions FOXO transcription factors activate 4E-BP expression amplifying the repression. Here we show that Drosophila FOXO binds the PDCD4 promoter and stimulates the transcription of PDCD4 in response to stress. We have shown previously that the 5' UTR of the Drosophila insulin-like receptor (dINR) supports cap-independent translation that is resistant to 4E-BP. Using hippuristanol, an eIF4A inhibitor, we find that translation of dINR UTR containing transcripts are also resistant to eIF4A inhibition. In addition, the murine insulin receptor and insulin-like growth factor receptor 5' UTRs support cap-independent translation and have a similar resistance to hippuristanol. This resistance to inhibition of eIF4E and eIF4A indicates a conserved strategy to allow translation of growth factor receptors under stress conditions.
Her2 activation mechanism reflects evolutionary preservation of asymmetric ectodomain dimers in the human EGFR family [eLife recent issues] 2013-07-16 12:17
The receptor tyrosine kinase Her2, an intensely pursued drug target, differs from other members of the EGFR family in that it does not bind EGF-like ligands, relying instead on heterodimerization with other (ligand-bound) EGFR-family receptors for activation. The structural basis for Her2 heterodimerization, however, remains poorly understood. The unexpected recent finding of asymmetric ectodomain dimer structures of Drosophila EGFR (dEGFR) suggests a possible structural basis for Her2 heterodimerization, but all available structures for dimers of human EGFR family ectodomains are symmetric. Here, we report results from long-timescale molecular dynamics simulations indicating that a single ligand is necessary and sufficient to stabilize the ectodomain interface of Her2 heterodimers, which assume an asymmetric conformation similar to that of dEGFR dimers. This structural parallelism suggests a dimerization mechanism that has been conserved in the evolution of the EGFR family from Drosophila to human.
An essential and NSF independent role for {alpha}-SNAP in store-operated calcium entry [eLife recent issues] 2013-07-16 12:17
Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca2+ sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE.
MYBL2 is a sub-haploinsufficient tumor suppressor gene in myeloid malignancy [eLife recent issues] 2013-07-16 12:17
A common deleted region (CDR) in both myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) affects the long arm of chromosome 20 and has been predicted to harbor a tumor suppressor gene. Here we show that MYBL2, a gene within the 20q CDR, is expressed at sharply reduced levels in CD34+ cells from most MDS cases (65%; n = 26), whether or not they harbor 20q abnormalities. In a murine competitive reconstitution model, Mybl2 knockdown by RNAi to 20–30% of normal levels in multipotent hematopoietic progenitors resulted in clonal dominance of these ‘sub-haploinsufficient’ cells, which was reflected in all blood cell lineages. By 6 months post-transplantation, the reconstituted mice had developed a clonal myeloproliferative/myelodysplastic disorder originating from the cells with aberrantly reduced Mybl2 expression. We conclude that downregulation of MYBL2 activity below levels predicted by classical haploinsufficiency underlies the clonal expansion of hematopoietic progenitors in a large fraction of human myeloid malignancies.
Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta [eLife recent issues] 2013-07-16 12:17
Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer’s, Parkinson’s, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer’s disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers.
Immune response is a personal matter [eLife recent issues] 2013-07-16 12:17
Changes in gene expression could be used to predict whether individuals will respond successfully to the influenza vaccine.
Lifting the veil on amyloid drug design [eLife recent issues] 2013-07-16 12:17
High resolution structures and computational methods have been used to identify compounds that prevent amyloid fibrils associated with Alzheimer’s disease from dissociating into toxic species.
A recipe for mediocrity and disaster, in five axioms [eLife recent issues] 2013-07-16 12:17
Biomedical research in the US will become unsustainable unless scientists and research institutions start to question certain assumptions they have long taken for granted.
Nutrient restriction enhances the proliferative potential of cells lacking the tumor suppressor PTEN in mitotic tissues [eLife recent issues] 2013-07-09 10:57
How single cells in a mitotic tissue progressively acquire hallmarks of cancer is poorly understood. We exploited mitotic recombination in developing Drosophila imaginal tissues to analyze the behavior of cells devoid of the tumor suppressor PTEN, a negative regulator of PI3K signaling, under varying nutritional conditions. Cells lacking PTEN strongly overproliferated specifically in nutrient restricted larvae. Although the PTEN mutant cells were sensitive to starvation, they successfully competed with neighboring cells by autonomous and non-autonomous mechanisms distinct from cell competition. The overgrowth was strictly dependent on the activity of the downstream components Akt/PKB and TORC1, and a reduction in amino acid uptake by reducing the levels of the amino acid transporter Slimfast caused clones of PTEN mutant cells to collapse. Our findings demonstrate how limiting nutritional conditions impact on cells lacking the tumor suppressor PTEN to cause hyperplastic overgrowth.
Apical targeting of the formin Diaphanous in Drosophila tubular epithelia [eLife recent issues] 2013-07-09 10:57
Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane.
Phosphorylation-mediated PTEN conformational closure and deactivation revealed with protein semisynthesis [eLife recent issues] 2013-07-09 10:57
The tumor suppressor PIP3 phosphatase PTEN is phosphorylated on four clustered Ser/Thr on its C-terminal tail (aa 380–385) and these phosphorylations are proposed to induce a reduction in PTEN’s plasma membrane recruitment. How these phosphorylations affect the structure and enzymatic function of PTEN is poorly understood. To gain insight into the mechanistic basis of PTEN regulation by phosphorylation, we generated semisynthetic site-specifically tetra-phosphorylated PTEN using expressed protein ligation. By employing a combination of biophysical and enzymatic approaches, we have found that purified tail-phosphorylated PTEN relative to its unphosphorylated counterpart shows reduced catalytic activity and membrane affinity and undergoes conformational compaction likely involving an intramolecular interaction between its C-tail and the C2 domain. Our results suggest that there is a competition between membrane phospholipids and PTEN phospho-tail for binding to the C2 domain. These findings reveal a key aspect of PTEN’s regulation and suggest pharmacologic approaches for direct PTEN activation.
TRiC's tricks inhibit huntingtin aggregation [eLife recent issues] 2013-07-09 10:57
In Huntington’s disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC’s inhibition of mhttQ51 aggregation in vitro.
Hypothemycin, a fungal natural product, identifies therapeutic targets in Trypanosoma brucei [eLife recent issues] 2013-07-09 10:57
Protein kinases are potentially attractive therapeutic targets for neglected parasitic diseases, including African trypanosomiasis caused by the protozoan, Trypanosoma brucei. How to prioritize T. brucei kinases and quantify their intracellular engagement by small-molecule inhibitors remain unsolved problems. Here, we combine chemoproteomics and RNA interference to interrogate trypanosome kinases bearing a Cys-Asp-Xaa-Gly motif (CDXG kinases). We discovered that hypothemycin, a fungal polyketide previously shown to covalently inactivate a subset of human CDXG kinases, kills T. brucei in culture and in infected mice. Quantitative chemoproteomic analysis with a hypothemycin-based probe revealed the relative sensitivity of endogenous CDXG kinases, including TbGSK3short and a previously uncharacterized kinase, TbCLK1. RNAi-mediated knockdown demonstrated that both kinases are essential, but only TbCLK1 is fully engaged by cytotoxic concentrations of hypothemycin in intact cells. Our study identifies TbCLK1 as a therapeutic target for African trypanosomiasis and establishes a new chemoproteomic tool for interrogating CDXG kinases in their native context.
Skill learning strengthens cortical representations of motor sequences [eLife recent issues] 2013-07-09 10:57
Motor-skill learning can be accompanied by both increases and decreases in brain activity. Increases may indicate neural recruitment, while decreases may imply that a region became unimportant or developed a more efficient representation of the skill. These overlapping mechanisms make interpreting learning-related changes of spatially averaged activity difficult. Here we show that motor-skill acquisition is associated with the emergence of highly distinguishable activity patterns for trained movement sequences, in the absence of average activity increases. During functional magnetic resonance imaging, participants produced either four trained or four untrained finger sequences. Using multivariate pattern analysis, both untrained and trained sequences could be discriminated in primary and secondary motor areas. However, trained sequences were classified more reliably, especially in the supplementary motor area. Our results indicate skill learning leads to the development of specialized neuronal circuits, which allow the execution of fast and accurate sequential movements without average increases in brain activity.
Closing in on a new treatment for sleeping sickness [eLife recent issues] 2013-07-09 10:57
A chemoproteomics approach has been employed to identify a kinase that could be used as a druggable target in efforts to develop new treatments for African sleeping sickness.
Direct single molecule measurement of TCR triggering by agonist pMHC in living primary T cells [eLife recent issues] 2013-07-03 09:29
T cells discriminate between self and foreign antigenic peptides, displayed on antigen presenting cell surfaces, via the TCR. While the molecular interactions between TCR and its ligands are well characterized in vitro, quantitative measurements of these interactions in living cells are required to accurately resolve the physical mechanisms of TCR signaling. We report direct single molecule measurements of TCR triggering by agonist pMHC in hybrid junctions between live primary T cells and supported lipid membranes. Every pMHC:TCR complex over the entire cell is tracked while simultaneously monitoring the local membrane recruitment of ZAP70, as a readout of TCR triggering. Mean dwell times for pMHC:TCR molecular binding of 5 and 54 s were measured for two different pMHC:TCR systems. Single molecule measurements of the pMHC:TCR:ZAP70 complex indicate that TCR triggering is stoichiometric with agonist pMHC in a 1:1 ratio. Thus any signal amplification must occur downstream of TCR triggering.
The genome sequence of the colonial chordate, Botryllus schlosseri [eLife recent issues] 2013-07-02 11:17
Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration.
A longitudinal study of Caenorhabditis elegans larvae reveals a novel locomotion switch, regulated by G{alpha}s signaling [eLife recent issues] 2013-07-02 11:17
Despite their simplicity, longitudinal studies of invertebrate models are rare. We thus sought to characterize behavioral trends of Caenorhabditis elegans, from the mid fourth larval stage through the mid young adult stage. We found that, outside of lethargus, animals exhibited abrupt switching between two distinct behavioral states: active wakefulness and quiet wakefulness. The durations of epochs of active wakefulness exhibited non-Poisson statistics. Increased Gαs signaling stabilized the active wakefulness state before, during and after lethargus. In contrast, decreased Gαs signaling, decreased neuropeptide release, or decreased CREB activity destabilized active wakefulness outside of, but not during, lethargus. Taken together, our findings support a model in which protein kinase A (PKA) stabilizes active wakefulness, at least in part through two of its downstream targets: neuropeptide release and CREB. However, during lethargus, when active wakefulness is strongly suppressed, the native role of PKA signaling in modulating locomotion and quiescence may be minor.
Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization [eLife recent issues] 2013-07-02 11:17
While host immune receptors detect pathogen-associated molecular patterns to activate immunity, pathogens attempt to deregulate host immunity through secreted effectors. Fungi employ LysM effectors to prevent recognition of cell wall-derived chitin by host immune receptors, although the mechanism to compete for chitin binding remained unclear. Structural analysis of the LysM effector Ecp6 of the fungal tomato pathogen Cladosporium fulvum reveals a novel mechanism for chitin binding, mediated by intrachain LysM dimerization, leading to a chitin-binding groove that is deeply buried in the effector protein. This composite binding site involves two of the three LysMs of Ecp6 and mediates chitin binding with ultra-high (pM) affinity. Intriguingly, the remaining singular LysM domain of Ecp6 binds chitin with low micromolar affinity but can nevertheless still perturb chitin-triggered immunity. Conceivably, the perturbation by this LysM domain is not established through chitin sequestration but possibly through interference with the host immune receptor complex.
Fluorescent sensors reporting the activity of ammonium transceptors in live cells [eLife recent issues] 2013-07-02 11:17
Ammonium serves as key nitrogen source and metabolic intermediate, yet excess causes toxicity. Ammonium uptake is mediated by ammonium transporters, whose regulation is poorly understood. While transport can easily be characterized in heterologous systems, measuring transporter activity in vivo remains challenging. Here we developed a simple assay for monitoring activity in vivo by inserting circularly-permutated GFP into conformation-sensitive positions of two plant and one yeast ammonium transceptors (‘AmTrac’ and ‘MepTrac’). Addition of ammonium to yeast cells expressing the sensors triggered concentration-dependent fluorescence intensity (FI) changes that strictly correlated with the activity of the transporter. Fluorescence-based activity sensors present a novel technology for monitoring the interaction of the transporters with their substrates, the activity of transporters and their regulation in vivo, which is particularly valuable in the context of analytes for which no radiotracers exist, as well as for cell-specific and subcellular transport processes that are otherwise difficult to track.
Getting to grips with ammonium [eLife recent issues] 2013-07-02 11:17
A fluorescent sensor that can monitor levels of extracellular ammonium has been made by using a fused green fluorescent protein to detect conformational changes in ammonium transport proteins.
Making science count in government [eLife recent issues] 2013-07-02 11:17
Science is an essential component of policy-making in most areas of government, but the scientific community does not always understand its role in this process.
Correction: The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine [eLife recent issues] 2013-07-02 11:17
Correction: Evolutionary principles of modular gene regulation in yeasts [eLife recent issues] 2013-07-02 11:17
When the time is ripe [eLife recent issues] 2013-06-25 11:12
The diverse effects of the plant hormone ethylene on development and growth are shaped by the actions of a master regulator of transcription, EIN3.
CAMKII and Calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16 [eLife recent issues] 2013-06-25 08:23
The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca2+/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin.
Rigid firing sequences undermine spatial memory codes in a neurodegenerative mouse model [eLife recent issues] 2013-06-25 08:23
Hippocampal neurons encode spatial memories by firing at specific locations. As the animal traverses a spatial trajectory, individual locations along the trajectory activate these neurons in a unique firing sequence, which yields a memory code representing the trajectory. How this type of memory code is altered in dementia-producing neurodegenerative disorders is unknown. Here we show that in transgenic rTg4510 mice, a model of tauopathies including Alzheimer's disease, hippocampal neurons did not fire at specific locations, yet displayed robust firing sequences as animals run along familiar or novel trajectories. The sequences seen on the trajectories also appeared during free exploration of open spaces. The spatially dissociated firing sequences suggest that hippocampal neurons in the transgenic mice are not primarily driven by external space but by internally generated brain activities. We propose that tau pathology and/or neurodegeneration renders hippocampal circuits overwhelmed by internal information and therefore prevents them from encoding spatial memories.
Arabidopsis plants perform arithmetic division to prevent starvation at night [eLife recent issues] 2013-06-25 08:23
Photosynthetic starch reserves that accumulate in Arabidopsis leaves during the day decrease approximately linearly with time at night to support metabolism and growth. We find that the rate of decrease is adjusted to accommodate variation in the time of onset of darkness and starch content, such that reserves last almost precisely until dawn. Generation of these dynamics therefore requires an arithmetic division computation between the starch content and expected time to dawn. We introduce two novel chemical kinetic models capable of implementing analog arithmetic division. Predictions from the models are successfully tested in plants perturbed by a night-time light period or by mutations in starch degradation pathways. Our experiments indicate which components of the starch degradation apparatus may be important for appropriate arithmetic division. Our results are potentially relevant for any biological system dependent on a food reserve for survival over a predictable time period.
Dismantling the Papez circuit for memory in rats [eLife recent issues] 2013-06-25 08:23
Over the last 50 years, anatomical models of memory have repeatedly highlighted the hippocampal inputs to the mammillary bodies via the postcommissural fornix. Such models downplay other projections to the mammillary bodies, leaving them largely ignored. The present study challenged this dominant view by removing, in rats, the two principal inputs reaching the mammillary bodies: the postcommissural fornix from the hippocampal formation and Gudden's ventral tegmental nucleus. The principal mammillary body output pathway, the mammillothalamic tract, was disconnected in a third group. Only mammillothalamic tract and Gudden's ventral tegmental nucleus lesions impaired behavioral tests of spatial working memory and, in particular, disrupted the use of extramaze spatial landmarks. The same lesions also produced widespread reductions in immediate-early gene (c-fos) expression in a network of memory-related regions, not seen after postcommissural fornix lesions. These findings are inconsistent with previous models of mammillary body function (those dominated by hippocampal inputs) and herald a new understanding of why specific diencephalic structures are vital for memory.
Evolutionary dynamics of cancer in response to targeted combination therapy [eLife recent issues] 2013-06-25 08:23
In solid tumors, targeted treatments can lead to dramatic regressions, but responses are often short-lived because resistant cancer cells arise. The major strategy proposed for overcoming resistance is combination therapy. We present a mathematical model describing the evolutionary dynamics of lesions in response to treatment. We first studied 20 melanoma patients receiving vemurafenib. We then applied our model to an independent set of pancreatic, colorectal, and melanoma cancer patients with metastatic disease. We find that dual therapy results in long-term disease control for most patients, if there are no single mutations that cause cross-resistance to both drugs; in patients with large disease burden, triple therapy is needed. We also find that simultaneous therapy with two drugs is much more effective than sequential therapy. Our results provide realistic expectations for the efficacy of new drug combinations and inform the design of trials for new cancer therapeutics.
Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1 [eLife recent issues] 2013-06-18 14:46
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life.
Evolutionary principles of modular gene regulation in yeasts [eLife recent issues] 2013-06-18 14:46
Divergence in gene regulation can play a major role in evolution. Here, we used a phylogenetic framework to measure mRNA profiles in 15 yeast species from the phylum Ascomycota and reconstruct the evolution of their modular regulatory programs along a time course of growth on glucose over 300 million years. We found that modules have diverged proportionally to phylogenetic distance, with prominent changes in gene regulation accompanying changes in lifestyle and ploidy, especially in carbon metabolism. Paralogs have significantly contributed to regulatory divergence, typically within a very short window from their duplication. Paralogs from a whole genome duplication (WGD) event have a uniquely substantial contribution that extends over a longer span. Similar patterns occur when considering the evolution of the heat shock regulatory program measured in eight of the species, suggesting that these are general evolutionary principles.
Histone demethylase Lsd1 represses hematopoietic stem and progenitor cell signatures during blood cell maturation [eLife recent issues] 2013-06-18 14:46
Here, we describe that lysine-specific demethylase 1 (Lsd1/KDM1a), which demethylates histone H3 on Lys4 or Lys9 (H3K4/K9), is an indispensible epigenetic governor of hematopoietic differentiation. Integrative genomic analysis, combining global occupancy of Lsd1, genome-wide analysis of its substrates H3K4 monomethylation and dimethylation, and gene expression profiling, reveals that Lsd1 represses hematopoietic stem and progenitor cell (HSPC) gene expression programs during hematopoietic differentiation. We found that Lsd1 acts at transcription start sites, as well as enhancer regions. Loss of Lsd1 was associated with increased H3K4me1 and H3K4me2 methylation on HSPC genes and gene derepression. Failure to fully silence HSPC genes compromised differentiation of hematopoietic stem cells as well as mature blood cell lineages. Collectively, our data indicate that Lsd1-mediated concurrent repression of enhancer and promoter activity of stem and progenitor cell genes is a pivotal epigenetic mechanism required for proper hematopoietic maturation.
The autoregulation of a eukaryotic DNA transposon [eLife recent issues] 2013-06-18 14:46
How do DNA transposons live in harmony with their hosts? Bacteria provide the only documented mechanisms for autoregulation, but these are incompatible with eukaryotic cell biology. Here we show that autoregulation of Hsmar1 operates during assembly of the transpososome and arises from the multimeric state of the transposase, mediated by a competition for binding sites. We explore the dynamics of a genomic invasion using a computer model, supported by in vitro and in vivo experiments, and show that amplification accelerates at first but then achieves a constant rate. The rate is proportional to the genome size and inversely proportional to transposase expression and its affinity for the transposon ends. Mariner transposons may therefore resist post-transcriptional silencing. Because regulation is an emergent property of the reaction it is resistant to selfish exploitation. The behavior of distantly related eukaryotic transposons is consistent with the same mechanism, which may therefore be widely applicable.
Condensin controls recruitment of RNA polymerase II to achieve nematode X-chromosome dosage compensation [eLife recent issues] 2013-06-18 14:46
The X-chromosome gene regulatory process called dosage compensation ensures that males (1X) and females (2X) express equal levels of X-chromosome transcripts. The mechanism in Caenorhabditis elegans has been elusive due to improperly annotated transcription start sites (TSSs). Here we define TSSs and the distribution of transcriptionally engaged RNA polymerase II (Pol II) genome-wide in wild-type and dosage-compensation-defective animals to dissect this regulatory mechanism. Our TSS-mapping strategy integrates GRO-seq, which tracks nascent transcription, with a new derivative of this method, called GRO-cap, which recovers nascent RNAs with 5' caps prior to their removal by co-transcriptional processing. Our analyses reveal that promoter-proximal pausing is rare, unlike in other metazoans, and promoters are unexpectedly far upstream from the 5' ends of mature mRNAs. We find that C. elegans equalizes X-chromosome expression between the sexes, to a level equivalent to autosomes, by reducing Pol II recruitment to promoters of hermaphrodite X-linked genes using a chromosome-restructuring condensin complex.
Yeast rises to the occasion [eLife recent issues] 2013-06-18 14:46
Genetic analyses of 15 species of yeast have shed new light on the divergence of gene regulation during evolution, with significant changes occurring after an event in which a whole genome was duplicated.
Keeping blood vessels out of sight [eLife recent issues] 2013-06-18 14:46
Researchers have identified a soluble receptor that prevents blood vessels forming in the outer retina—a process that can lead to blindness—by sequestering vascular endothelial growth factor.
The early days of late blight [eLife recent issues] 2013-06-18 14:46
Large-scale DNA sequencing of samples of foliage collected in the 19th century from plants infected with late blight has shown that the potato famines of the 1840s were triggered by a single clonal lineage of Phytophthora infestans, called HERB-1, which persisted for at least 50 years.
The lasting influence of LSD1 in the blood [eLife recent issues] 2013-06-18 14:46
An enzyme called LSD1 that controls the development of blood cells by manipulating gene expression in progenitor cells could be a therapeutic target for leukemia.
Viral genome structures are optimal for capsid assembly [eLife recent issues] 2013-06-14 10:13
Understanding how virus capsids assemble around their nucleic acid (NA) genomes could promote efforts to block viral propagation or to reengineer capsids for gene therapy applications. We develop a coarse-grained model of capsid proteins and NAs with which we investigate assembly dynamics and thermodynamics. In contrast to recent theoretical models, we find that capsids spontaneously ‘overcharge’; that is, the negative charge of the NA exceeds the positive charge on capsid. When applied to specific viruses, the optimal NA lengths closely correspond to the natural genome lengths. Calculations based on linear polyelectrolytes rather than base-paired NAs underpredict the optimal length, demonstrating the importance of NA structure to capsid assembly. These results suggest that electrostatics, excluded volume, and NA tertiary structure are sufficient to predict assembly thermodynamics and that the ability of viruses to selectively encapsidate their genomic NAs can be explained, at least in part, on a thermodynamic basis.
Correction: Passive and active DNA methylation and the interplay with genetic variation in gene regulation [eLife recent issues] 2013-06-14 10:13
The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability [eLife recent issues] 2013-06-11 11:02
Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo.
miR-124 controls male reproductive success in Drosophila [eLife recent issues] 2013-06-11 11:02
Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male–male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila.
Temporal transcriptional response to ethylene gas drives growth hormone cross-regulation in Arabidopsis [eLife recent issues] 2013-06-11 11:02
The gaseous plant hormone ethylene regulates a multitude of growth and developmental processes. How the numerous growth control pathways are coordinated by the ethylene transcriptional response remains elusive. We characterized the dynamic ethylene transcriptional response by identifying targets of the master regulator of the ethylene signaling pathway, ETHYLENE INSENSITIVE3 (EIN3), using chromatin immunoprecipitation sequencing and transcript sequencing during a timecourse of ethylene treatment. Ethylene-induced transcription occurs in temporal waves regulated by EIN3, suggesting distinct layers of transcriptional control. EIN3 binding was found to modulate a multitude of downstream transcriptional cascades, including a major feedback regulatory circuitry of the ethylene signaling pathway, as well as integrating numerous connections between most of the hormone mediated growth response pathways. These findings provide direct evidence linking each of the major plant growth and development networks in novel ways.
Intraflagellar transport drives flagellar surface motility [eLife recent issues] 2013-06-11 11:02
The assembly and maintenance of all cilia and flagella require intraflagellar transport (IFT) along the axoneme. IFT has been implicated in sensory and motile ciliary functions, but the mechanisms of this relationship remain unclear. Here, we used Chlamydomonas flagellar surface motility (FSM) as a model to test whether IFT provides force for gliding of cells across solid surfaces. We show that IFT trains are coupled to flagellar membrane glycoproteins (FMGs) in a Ca2+-dependent manner. IFT trains transiently pause through surface adhesion of their FMG cargos, and dynein-1b motors pull the cell towards the distal tip of the axoneme. Each train is transported by at least four motors, with only one type of motor active at a time. Our results demonstrate the mechanism of Chlamydomonas gliding motility and suggest that IFT plays a major role in adhesion-induced ciliary signaling pathways.
Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors [eLife recent issues] 2013-06-11 11:02
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery.
Hopping rim to rim through the Golgi [eLife recent issues] 2013-06-11 11:02
A novel approach based on tracking the fate of proteins that become ‘stapled’ to the walls of the Golgi yields insights into the long-sought mechanism of transport through this organelle.
Rad51, friend or foe? [eLife recent issues] 2013-06-11 11:02
A protein long recognized for its role in DNA repair has now paradoxically been implicated in DNA damage.
Correction: Feeding-induced rearrangement of green leaf volatiles reduces moth oviposition [eLife recent issues] 2013-06-11 11:02
Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1 [eLife recent issues] 2013-06-04 12:02
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
Passive and active DNA methylation and the interplay with genetic variation in gene regulation [eLife recent issues] 2013-06-04 12:02
DNA methylation is an essential epigenetic mark whose role in gene regulation and its dependency on genomic sequence and environment are not fully understood. In this study we provide novel insights into the mechanistic relationships between genetic variation, DNA methylation and transcriptome sequencing data in three different cell-types of the GenCord human population cohort. We find that the association between DNA methylation and gene expression variation among individuals are likely due to different mechanisms from those establishing methylation-expression patterns during differentiation. Furthermore, cell-type differential DNA methylation may delineate a platform in which local inter-individual changes may respond to or act in gene regulation. We show that unlike genetic regulatory variation, DNA methylation alone does not significantly drive allele specific expression. Finally, inferred mechanistic relationships using genetic variation as well as correlations with TF abundance reveal both a passive and active role of DNA methylation to regulatory interactions influencing gene expression.
Stapled Golgi cisternae remain in place as cargo passes through the stack [eLife recent issues] 2013-06-04 12:02
We have designed a membrane ‘staple’, which consists of membrane-anchored repeats of the trans-aggregating FM domain that face the lumen of the secretory pathway. In the presence of the disaggregating drug these proteins transit the secretory pathway. When the drug is removed these proteins form electron-dense plaques which we term staples. Unexpectedly, when initially positioned within the cis-Golgi, staples remained at the cis face of the Golgi even after many hours. By contrast, soluble FM-aggregates transited the Golgi. Staples and soluble aggregates placed in cis-Golgi cisternae therefore have different fates. Whereas the membrane staples are located in the flattened, stacked central regions of the cisternae, the soluble aggregates are in the dilated rims. This suggests that while the cisternae are static on the time scale of protein traffic, the dilated rims are mobile and progress in the cis -> trans direction via a mechanism that we term ‘Rim Progression’.
MCU encodes the pore conducting mitochondrial calcium currents [eLife recent issues] 2013-06-04 12:02
Mitochondrial calcium (Ca2+) import is a well-described phenomenon regulating cell survival and ATP production. Of multiple pathways allowing such entry, the mitochondrial Ca2+ uniporter is a highly Ca2+-selective channel complex encoded by several recently-discovered genes. However, the identity of the pore-forming subunit remains to be established, since knockdown of all the candidate uniporter genes inhibit Ca2+ uptake in imaging assays, and reconstitution experiments have been equivocal. To definitively identify the channel, we use whole-mitoplast voltage-clamping, the technique that originally established the uniporter as a Ca2+ channel. We show that RNAi-mediated knockdown of the mitochondrial calcium uniporter (MCU) gene reduces mitochondrial Ca2+ current (IMiCa), whereas overexpression increases it. Additionally, a classic feature of IMiCa, its sensitivity to ruthenium red inhibition, can be abolished by a point mutation in the putative pore domain without altering current magnitude. These analyses establish that MCU encodes the pore-forming subunit of the uniporter channel.
Sequence-dependent base pair stepping dynamics in XPD helicase unwinding [eLife recent issues] 2013-05-28 15:18
Helicases couple the chemical energy of ATP hydrolysis to directional translocation along nucleic acids and transient duplex separation. Understanding helicase mechanism requires that the basic physicochemical process of base pair separation be understood. This necessitates monitoring helicase activity directly, at high spatio-temporal resolution. Using optical tweezers with single base pair (bp) resolution, we analyzed DNA unwinding by XPD helicase, a Superfamily 2 (SF2) DNA helicase involved in DNA repair and transcription initiation. We show that monomeric XPD unwinds duplex DNA in 1-bp steps, yet exhibits frequent backsteps and undergoes conformational transitions manifested in 5-bp backward and forward steps. Quantifying the sequence dependence of XPD stepping dynamics with near base pair resolution, we provide the strongest and most direct evidence thus far that forward, single-base pair stepping of a helicase utilizes the spontaneous opening of the duplex. The proposed unwinding mechanism may be a universal feature of DNA helicases that move along DNA phosphodiester backbones.
Decoding the neural mechanisms of human tool use [eLife recent issues] 2013-05-28 15:18
Sophisticated tool use is a defining characteristic of the primate species but how is it supported by the brain, particularly the human brain? Here we show, using functional MRI and pattern classification methods, that tool use is subserved by multiple distributed action-centred neural representations that are both shared with and distinct from those of the hand. In areas of frontoparietal cortex we found a common representation for planned hand- and tool-related actions. In contrast, in parietal and occipitotemporal regions implicated in hand actions and body perception we found that coding remained selectively linked to upcoming actions of the hand whereas in parietal and occipitotemporal regions implicated in tool-related processing the coding remained selectively linked to upcoming actions of the tool. The highly specialized and hierarchical nature of this coding suggests that hand- and tool-related actions are represented separately at earlier levels of sensorimotor processing before becoming integrated in frontoparietal cortex.
Pharmacological brake-release of mRNA translation enhances cognitive memory [eLife recent issues] 2013-05-28 15:18
Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.
TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells [eLife recent issues] 2013-05-28 15:18
Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca2+-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca2+ signal. ATP-induced MUC5AC secretion depended strongly on Ca2+ influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca2+ entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca2+ and by inhibition of the Na+/Ca2+ exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na+ entry to promote Ca2+ uptake via an NCX to trigger MUC5AC secretion.
The rise and fall of the Phytophthora infestans lineage that triggered the Irish potato famine [eLife recent issues] 2013-05-28 15:18
Phytophthora infestans, the cause of potato late blight, is infamous for having triggered the Irish Great Famine in the 1840s. Until the late 1970s, P. infestans diversity outside of its Mexican center of origin was low, and one scenario held that a single strain, US-1, had dominated the global population for 150 years; this was later challenged based on DNA analysis of historical herbarium specimens. We have compared the genomes of 11 herbarium and 15 modern strains. We conclude that the 19th century epidemic was caused by a unique genotype, HERB-1, that persisted for over 50 years. HERB-1 is distinct from all examined modern strains, but it is a close relative of US-1, which replaced it outside of Mexico in the 20th century. We propose that HERB-1 and US-1 emerged from a metapopulation that was established in the early 1800s outside of the species' center of diversity.
Building for the future [eLife recent issues] 2013-05-28 15:18
As the staff of the MRC Laboratory of Molecular Biology settle into their new building in Cambridge, its director Hugh Pelham explains the challenges of living up to its prestigious past.
Watching the brain in action [eLife recent issues] 2013-05-28 15:18
Functional magnetic resonance imaging has been used to identify the different networks in the brain that underpin the use of tools by humans.
Less translational control, more memory [eLife recent issues] 2013-05-28 15:18
A small molecule can enhance the memories of rats and mice by blocking the integrated stress response in these animals.
MicroRNA-146a acts as a guardian of the quality and longevity of hematopoietic stem cells in mice [eLife recent issues] 2013-05-21 12:07
During inflammation and infection, hematopoietic stem and progenitor cells are stimulated to proliferate and differentiate into mature immune cells, especially of the myeloid lineage. MicroRNA-146a (miR-146a) is a critical negative regulator of inflammation. Deletion of miR-146a produces effects that appear as dysregulated inflammatory hematopoiesis, leading to a decline in the number and quality of hematopoietic stem cells (HSCs), excessive myeloproliferation, and, ultimately, to HSC exhaustion and hematopoietic neoplasms. At the cellular level, the defects are attributable to both an intrinsic problem in the miR-146a–deficient HSCs and extrinsic effects of lymphocytes and nonhematopoietic cells. At the molecular level, this involves a molecular axis consisting of miR-146a, signaling protein TRAF6, transcriptional factor NF-B, and cytokine IL-6. This study has identified miR-146a to be a critical regulator of HSC homeostasis during chronic inflammation in mice and provided a molecular connection between chronic inflammation and the development of bone marrow failure and myeloproliferative neoplasms.
Structure of a pore-blocking toxin in complex with a eukaryotic voltage-dependent K+ channel [eLife recent issues] 2013-05-21 12:07
Pore-blocking toxins inhibit voltage-dependent K+ channels (Kv channels) by plugging the ion-conduction pathway. We have solved the crystal structure of paddle chimera, a Kv channel in complex with charybdotoxin (CTX), a pore-blocking toxin. The toxin binds to the extracellular pore entryway without producing discernable alteration of the selectivity filter structure and is oriented to project its Lys27 into the pore. The most extracellular K+ binding site (S1) is devoid of K+ electron-density when wild-type CTX is bound, but K+ density is present to some extent in a Lys27Met mutant. In crystals with Cs+ replacing K+, S1 electron-density is present even in the presence of Lys27, a finding compatible with the differential effects of Cs+ vs K+ on CTX affinity for the channel. Together, these results show that CTX binds to a K+ channel in a lock and key manner and interacts directly with conducting ions inside the selectivity filter.
Predicting mosquito infection from Plasmodium falciparum gametocyte density and estimating the reservoir of infection [eLife recent issues] 2013-05-21 12:07
Transmission reduction is a key component of global efforts to control and eliminate malaria; yet, it is unclear how the density of transmission stages (gametocytes) influences infection (proportion of mosquitoes infected). Human to mosquito transmission was assessed using 171 direct mosquito feeding assays conducted in Burkina Faso and Kenya. Plasmodium falciparum infects Anopheles gambiae efficiently at low densities (4% mosquitoes at 1/µl blood), although substantially more (>200/µl) are required to increase infection further. In a site in Burkina Faso, children harbour more gametocytes than adults though the non-linear relationship between gametocyte density and mosquito infection means that (per person) they only contribute slightly more to transmission. This method can be used to determine the reservoir of infection in different endemic settings. Interventions reducing gametocyte density need to be highly effective in order to halt human–mosquito transmission, although their use can be optimised by targeting those contributing the most to transmission.
The scorpion toxin and the potassium channel [eLife recent issues] 2013-05-21 12:07
The structure of a complex containing a toxin bound to a potassium ion channel has been solved for the first time, revealing how scorpions have designed toxins that can recognize and target the filter that controls the movement of potassium ions through these channels.
A novel sphingolipid-TORC1 pathway critically promotes postembryonic development in Caenorhabditis elegans [eLife recent issues] 2013-05-21 12:07
Regulation of animal development in response to nutritional cues is an intensely studied problem related to disease and aging. While extensive studies indicated roles of the Target of Rapamycin (TOR) in sensing certain nutrients for controlling growth and metabolism, the roles of fatty acids and lipids in TOR-involved nutrient/food responses are obscure. Caenorhabditis elegans halts postembryonic growth and development shortly after hatching in response to monomethyl branched-chain fatty acid (mmBCFA) deficiency. Here, we report that an mmBCFA-derived sphingolipid, d17iso-glucosylceramide, is a critical metabolite in regulating growth and development. Further analysis indicated that this lipid function is mediated by TORC1 and antagonized by the NPRL-2/3 complex in the intestine. Strikingly, the essential lipid function is bypassed by activating TORC1 or inhibiting NPRL-2/3. Our findings uncover a novel lipid-TORC1 signaling pathway that coordinates nutrient and metabolic status with growth and development, advancing our understanding of the physiological roles of mmBCFAs, ceramides, and TOR.
Reforming research assessment [eLife recent issues] 2013-05-16 14:02
It is time for the research community to rethink how the outputs of scientific research are evaluated and, as the San Francisco Declaration on Research Assessment makes clear, this should involve replacing the journal impact factor with a broad range of more meaningful approaches.
EBI2-mediated bridging channel positioning supports splenic dendritic cell homeostasis and particulate antigen capture [eLife recent issues] 2013-05-14 09:46
Splenic dendritic cells (DCs) present blood-borne antigens to lymphocytes to promote T cell and antibody responses. The cues involved in positioning DCs in areas of antigen exposure in the spleen are undefined. Here we show that CD4+ DCs highly express EBI2 and migrate to its oxysterol ligand, 7α,25-OHC. In mice lacking EBI2 or the enzymes needed for generating normal distributions of 7α,25-OHC, CD4+ DCs are reduced in frequency and the remaining cells fail to situate in marginal zone bridging channels. The CD4+ DC deficiency can be rescued by LTβR agonism. EBI2-mediated positioning in bridging channels promotes DC encounter with blood-borne particulate antigen. Upon exposure to antigen, CD4+ DCs move rapidly to the T-B zone interface and promote induction of helper T cell and antibody responses. These findings establish an essential role for EBI2 in CD4+ DC positioning and homeostasis and in facilitating capture and presentation of blood-borne particulate antigens.
Feeding-induced rearrangement of green leaf volatiles reduces moth oviposition [eLife recent issues] 2013-05-14 08:56
The ability to decrypt volatile plant signals is essential if herbivorous insects are to optimize their choice of host plants for their offspring. Green leaf volatiles (GLVs) constitute a widespread group of defensive plant volatiles that convey a herbivory-specific message via their isomeric composition: feeding of the tobacco hornworm Manduca sexta converts (Z)-3- to (E)-2-GLVs thereby attracting predatory insects. Here we show that this isomer-coded message is monitored by ovipositing M. sexta females. We detected the isomeric shift in the host plant Datura wrightii and performed functional imaging in the primary olfactory center of M. sexta females with GLV structural isomers. We identified two isomer-specific regions responding to either (Z)-3- or (E)-2-hexenyl acetate. Field experiments demonstrated that ovipositing Manduca moths preferred (Z)-3-perfumed D. wrightii over (E)-2-perfumed plants. These results show that (E)-2-GLVs and/or specific (Z)-3/(E)-2-ratios provide information regarding host plant attack by conspecifics that ovipositing hawkmoths use for host plant selection.
ER-associated mitochondrial division links the distribution of mitochondria and mitochondrial DNA in yeast [eLife recent issues] 2013-05-14 08:56
Mitochondrial division is important for mitochondrial distribution and function. Recent data have demonstrated that ER–mitochondria contacts mark mitochondrial division sites, but the molecular basis and functions of these contacts are not understood. Here we show that in yeast, the ER–mitochondria tethering complex, ERMES, and the highly conserved Miro GTPase, Gem1, are spatially and functionally linked to ER-associated mitochondrial division. Gem1 acts as a negative regulator of ER–mitochondria contacts, an activity required for the spatial resolution and distribution of newly generated mitochondrial tips following division. Previous data have demonstrated that ERMES localizes with a subset of actively replicating mitochondrial nucleoids. We show that mitochondrial division is spatially linked to nucleoids and that a majority of these nucleoids segregate prior to division, resulting in their distribution into newly generated tips in the mitochondrial network. Thus, we postulate that ER-associated division serves to link the distribution of mitochondria and mitochondrial nucleoids in cells.
Controlling gain one photon at a time [eLife recent issues] 2013-05-14 08:56
Adaptation is a salient property of sensory processing. All adaptational or gain control mechanisms face the challenge of obtaining a reliable estimate of the property of the input to be adapted to and obtaining this estimate sufficiently rapidly to be useful. Here, we explore how the primate retina balances the need to change gain rapidly and reliably when photons arrive rarely at individual rod photoreceptors. We find that the weakest backgrounds that decrease the gain of the retinal output signals are similar to those that increase human behavioral threshold, and identify a novel site of gain control in the retinal circuitry. Thus, surprisingly, the gain of retinal signals begins to decrease essentially as soon as background lights are detectable; under these conditions, gain control does not rely on a highly averaged estimate of the photon count, but instead signals from individual photon absorptions trigger changes in gain.
Stability-mediated epistasis constrains the evolution of an influenza protein [eLife recent issues] 2013-05-14 08:56
John Maynard Smith compared protein evolution to the game where one word is converted into another a single letter at a time, with the constraint that all intermediates are words: WORD->WORE->GORE->GONE->GENE. In this analogy, epistasis constrains evolution, with some mutations tolerated only after the occurrence of others. To test whether epistasis similarly constrains actual protein evolution, we created all intermediates along a 39-mutation evolutionary trajectory of influenza nucleoprotein, and also introduced each mutation individually into the parent. Several mutations were deleterious to the parent despite becoming fixed during evolution without negative impact. These mutations were destabilizing, and were preceded or accompanied by stabilizing mutations that alleviated their adverse effects. The constrained mutations occurred at sites enriched in T-cell epitopes, suggesting they promote viral immune escape. Our results paint a coherent portrait of epistasis during nucleoprotein evolution, with stabilizing mutations permitting otherwise inaccessible destabilizing mutations which are sometimes of adaptive value.
Make or break for mitochondria [eLife recent issues] 2013-05-14 08:56
Ensuring that mitochondrial DNA is successfully divided between daughter mitochondria involves a complex series of interactions with the endoplasmic reticulum and a variety of enzymes.
Influenza evolution navigates stability valleys [eLife recent issues] 2013-05-14 08:56
By reconstructing how an influenza protein collected in 1968 might have evolved into one collected in 2007, researchers have obtained new insights into the interactions between genetic mutations.
A role for PVRL4-driven cell-cell interactions in tumorigenesis [eLife recent issues] 2013-04-30 11:56
During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer.
Accurate timekeeping is controlled by a cycling activator in Arabidopsis [eLife recent issues] 2013-04-30 08:01
Transcriptional feedback loops are key to circadian clock function in many organisms. Current models of the Arabidopsis circadian network consist of several coupled feedback loops composed almost exclusively of transcriptional repressors. Indeed, a central regulatory mechanism is the repression of evening-phased clock genes via the binding of morning-phased Myb-like repressors to evening element (EE) promoter motifs. We now demonstrate that a related Myb-like protein, REVEILLE8 (RVE8), is a direct transcriptional activator of EE-containing clock and output genes. Loss of RVE8 and its close homologs causes a delay and reduction in levels of evening-phased clock gene transcripts and significant lengthening of clock pace. Our data suggest a substantially revised model of the circadian oscillator, with a clock-regulated activator essential both for clock progression and control of clock outputs. Further, our work suggests that the plant clock consists of a highly interconnected, complex regulatory network rather than of coupled morning and evening feedback loops.
Contributions of mast cells and vasoactive products, leukotrienes and chymase, to dengue virus-induced vascular leakage [eLife recent issues] 2013-04-30 08:01
Dengue Virus (DENV), a flavivirus spread by mosquito vectors, can cause vascular leakage and hemorrhaging. However, the processes that underlie increased vascular permeability and pathological plasma leakage during viral hemorrhagic fevers are largely unknown. Mast cells (MCs) are activated in vivo during DENV infection, and we show that this elevates systemic levels of their vasoactive products, including chymase, and promotes vascular leakage. Treatment of infected animals with MC-stabilizing drugs or a leukotriene receptor antagonist restores vascular integrity during experimental DENV infection. Validation of these findings using human clinical samples revealed a direct correlation between MC activation and DENV disease severity. In humans, the MC-specific product, chymase, is a predictive biomarker distinguishing dengue fever (DF) and dengue hemorrhagic fever (DHF). Additionally, our findings reveal MCs as potential therapeutic targets to prevent DENV-induced vasculopathy, suggesting MC-stabilizing drugs should be evaluated for their effectiveness in improving disease outcomes during viral hemorrhagic fevers.
Native {alpha}-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2 [eLife recent issues] 2013-04-30 08:01
α-Synuclein is a presynaptic protein that is implicated in Parkinson's and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinson's disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself.
Unmasking the role of mast cells in dengue [eLife recent issues] 2013-04-30 08:01
Immune cells called mast cells can hinder rather than help the body's response to dengue virus, which suggests that mast cell products could be used as biomarkers to identify severe forms of the disease.
Time for a change [eLife recent issues] 2013-04-30 08:01
The circadian clock of Arabidopsis, a popular model organism for plants, is more complex than expected, with negative feedback loops based on the repression of gene expression having a less exclusive role than previously thought.
The eLife approach to peer review [eLife recent issues] 2013-04-30 08:01
All editorial decisions at eLife are taken by working scientists in a process that emphasizes fairness, speed and transparency.
Ovulation in Drosophila is controlled by secretory cells of the female reproductive tract [eLife recent issues] 2013-04-16 13:11
How oocytes are transferred into an oviduct with a receptive environment remains poorly known. We found that glands of the Drosophila female reproductive tract, spermathecae and/or parovaria, are required for ovulation and to promote sperm storage. Reducing total secretory cell number by interferring with Notch signaling during development blocked ovulation. Knocking down expression after adult eclosion of the nuclear hormone receptor Hr39, a master regulator of gland development, slowed ovulation and blocked sperm storage. However, ovulation (but not sperm storage) continued when only canonical protein secretion was compromised in adult glands. Our results imply that proteins secreted during adulthood by the canonical secretory pathway from female reproductive glands are needed to store sperm, while a non-canonical glandular secretion stimulates ovulation. Our results suggest that the reproductive tract signals to the ovary using glandular secretions, and that this pathway has been conserved during evolution.
Evolution, ovulation and cancer [eLife recent issues] 2013-04-16 13:11
Secretions by epithelial cells of the fallopian tube regulate ovulation through conserved pathways, which means that experiments on flies might provide insights into the human reproductive system and, possibly, ovarian cancer.
Rapid localized spread and immunologic containment define Herpes simplex virus-2 reactivation in the human genital tract [eLife recent issues] 2013-04-16 10:06
Herpes simplex virus-2 (HSV-2) is shed episodically, leading to occasional genital ulcers and efficient transmission. The biology explaining highly variable shedding patterns, in an infected person over time, is poorly understood. We sampled the genital tract for HSV DNA at several time intervals and concurrently at multiple sites, and derived a spatial mathematical model to characterize dynamics of HSV-2 reactivation. The model reproduced heterogeneity in shedding episode duration and viral production, and predicted rapid early viral expansion, rapid late decay, and wide spatial dispersion of HSV replication during episodes. In simulations, HSV-2 spread locally within single ulcers to thousands of epithelial cells in <12 hr, but host immune responses eliminated infected cells in <24 hr; secondary ulcers formed following spatial propagation of cell-free HSV-2, allowing for episode prolongation. We conclude that HSV-2 infection is characterized by extremely rapid virological growth and containment at multiple contemporaneous sites within genital epithelium.
Acute stress enhances adult rat hippocampal neurogenesis and activation of newborn neurons via secreted astrocytic FGF2 [eLife recent issues] 2013-04-16 10:06
Stress is a potent modulator of the mammalian brain. The highly conserved stress hormone response influences many brain regions, particularly the hippocampus, a region important for memory function. The effect of acute stress on the unique population of adult neural stem/progenitor cells (NPCs) that resides in the adult hippocampus is unclear. We found that acute stress increased hippocampal cell proliferation and astrocytic fibroblast growth factor 2 (FGF2) expression. The effect of acute stress occurred independent of basolateral amygdala neural input and was mimicked by treating isolated NPCs with conditioned media from corticosterone-treated primary astrocytes. Neutralization of FGF2 revealed that astrocyte-secreted FGF2 mediated stress-hormone-induced NPC proliferation. 2 weeks, but not 2 days, after acute stress, rats also showed enhanced fear extinction memory coincident with enhanced activation of newborn neurons. Our findings suggest a beneficial role for brief stress on the hippocampus and improve understanding of the adaptive capacity of the brain.
Cohabiting family members share microbiota with one another and with their dogs [eLife recent issues] 2013-04-16 10:06
Human-associated microbial communities vary across individuals: possible contributing factors include (genetic) relatedness, diet, and age. However, our surroundings, including individuals with whom we interact, also likely shape our microbial communities. To quantify this microbial exchange, we surveyed fecal, oral, and skin microbiota from 60 families (spousal units with children, dogs, both, or neither). Household members, particularly couples, shared more of their microbiota than individuals from different households, with stronger effects of co-habitation on skin than oral or fecal microbiota. Dog ownership significantly increased the shared skin microbiota in cohabiting adults, and dog-owning adults shared more ‘skin’ microbiota with their own dogs than with other dogs. Although the degree to which these shared microbes have a true niche on the human body, vs transient detection after direct contact, is unknown, these results suggest that direct and frequent contact with our cohabitants may significantly shape the composition of our microbial communities.
The ART-Rsp5 ubiquitin ligase network comprises a plasma membrane quality control system that protects yeast cells from proteotoxic stress [eLife recent issues] 2013-04-16 10:06
Secretory cargo that cannot fold properly in the ER are selectively targeted for removal by a well-studied ER-associated degradation pathway, or ERAD. In contrast, very little is known about post-ER quality control mechanisms for damaged or misfolded integral membrane proteins. Here we describe a quality control function of the Rsp5-ART ubiquitin ligase adaptor network that functions to protect plasma membrane (PM) integrity. Failure to mediate this protective response during heat stress leads to toxic accumulation of misfolded integral membrane proteins at the cell surface, which causes loss of PM integrity and cell death. Thus, the Rsp5-ART network comprises a PM quality control system that works together with sequential quality control pathways in the ER and Golgi to (i) target the degradation of proteins that have exceeded their functional lifetime due to damage and/or misfolding and (ii) limit the toxic accumulation of specific proteins at the cell surface during proteotoxic stress.
DNA deaminases induce break-associated mutation showers with implication of APOBEC3B and 3A in breast cancer kataegis [eLife recent issues] 2013-04-16 10:06
Breast cancer genomes have revealed a novel form of mutation showers (kataegis) in which multiple same-strand substitutions at C:G pairs spaced one to several hundred nucleotides apart are clustered over kilobase-sized regions, often associated with sites of DNA rearrangement. We show kataegis can result from AID/APOBEC-catalysed cytidine deamination in the vicinity of DNA breaks, likely through action on single-stranded DNA exposed during resection. Cancer-like kataegis can be recapitulated by expression of AID/APOBEC family deaminases in yeast where it largely depends on uracil excision, which generates an abasic site for strand breakage. Localized kataegis can also be nucleated by an I-SceI-induced break. Genome-wide patterns of APOBEC3-catalyzed deamination in yeast reveal APOBEC3B and 3A as the deaminases whose mutational signatures are most similar to those of breast cancer kataegic mutations. Together with expression and functional assays, the results implicate APOBEC3B/A in breast cancer hypermutation and give insight into the mechanism of kataegis.
Luck, jobs and learning [eLife recent issues] 2013-04-16 10:06
Eve Marder believes that many of the most important events in our lives, both personal and professional, depend to some degree on luck or chance.
SEC24A deficiency lowers plasma cholesterol through reduced PCSK9 secretion [eLife recent issues] 2013-04-09 08:01
The secretory pathway of eukaryotic cells packages cargo proteins into COPII-coated vesicles for transport from the endoplasmic reticulum (ER) to the Golgi. We now report that complete genetic deficiency for the COPII component SEC24A is compatible with normal survival and development in the mouse, despite the fundamental role of SEC24 in COPII vesicle formation and cargo recruitment. However, these animals exhibit markedly reduced plasma cholesterol, with mutations in Apoe and Ldlr epistatic to Sec24a, suggesting a receptor-mediated lipoprotein clearance mechanism. Consistent with these data, hepatic LDLR levels are up-regulated in SEC24A-deficient cells as a consequence of specific dependence of PCSK9, a negative regulator of LDLR, on SEC24A for efficient exit from the ER. Our findings also identify partial overlap in cargo selectivity between SEC24A and SEC24B, suggesting a previously unappreciated heterogeneity in the recruitment of secretory proteins to the COPII vesicles that extends to soluble as well as trans-membrane cargoes.
Overcoming mutation-based resistance to antiandrogens with rational drug design [eLife recent issues] 2013-04-09 08:01
The second-generation antiandrogen enzalutamide was recently approved for patients with castration-resistant prostate cancer. Despite its success, the duration of response is often limited. For previous antiandrogens, one mechanism of resistance is mutation of the androgen receptor (AR). To prospectively identify AR mutations that might confer resistance to enzalutamide, we performed a reporter-based mutagenesis screen and identified a novel mutation, F876L, which converted enzalutamide into an AR agonist. Ectopic expression of AR F876L rescued the growth inhibition of enzalutamide treatment. Molecular dynamics simulations performed on antiandrogen–AR complexes suggested a mechanism by which the F876L substitution alleviates antagonism through repositioning of the coactivator recruiting helix 12. This model then provided the rationale for a focused chemical screen which, based on existing antiandrogen scaffolds, identified three novel compounds that effectively antagonized AR F876L (and AR WT) to suppress the growth of prostate cancer cells resistant to enzalutamide.
Shedding new light on circadian clocks [eLife recent issues] 2013-04-09 08:01
Using a clever combination of genetic and biochemical tools researchers have shown that a transcription factor called USF1 has a central role in determining how mutations of the Clock gene manifest themselves in the behaviour of different mouse strains.
Opening up new fronts in the fight against cholesterol [eLife recent issues] 2013-04-09 08:01
An unexpected connection between a secretory protein called PCSK9 and Sec24A, a well known protein-transport protein, could lead to the development of novel treatments for patients with high levels of low-density lipoproteins in their blood.
Designer antiandrogens join the race against drug resistance [eLife recent issues] 2013-04-09 08:01
By using in silico models of the complexes formed by analogues of a cancer drug and its receptor, it may be possible to strategically redesign existing drugs and win the race against mutations that lead to drug resistance in prostate cancer.
Usf1, a suppressor of the circadian Clock mutant, reveals the nature of the DNA-binding of the CLOCK:BMAL1 complex in mice [eLife recent issues] 2013-04-09 08:01
Genetic and molecular approaches have been critical for elucidating the mechanism of the mammalian circadian clock. Here, we demonstrate that the Clock19 mutant behavioral phenotype is significantly modified by mouse strain genetic background. We map a suppressor of the Clock19 mutation to a ~900 kb interval on mouse chromosome 1 and identify the transcription factor, Usf1, as the responsible gene. A SNP in the promoter of Usf1 causes elevation of its transcript and protein in strains that suppress the Clock mutant phenotype. USF1 competes with the CLOCK:BMAL1 complex for binding to E-box sites in target genes. Saturation binding experiments demonstrate reduced affinity of the CLOCK19:BMAL1 complex for E-box sites, thereby permitting increased USF1 occupancy on a genome-wide basis. We propose that USF1 is an important modulator of molecular and behavioral circadian rhythms in mammals.
Stepwise assembly of the human replicative polymerase holoenzyme [eLife recent issues] 2013-04-02 08:16
In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (pol), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication.
Improved use of a public good selects for the evolution of undifferentiated multicellularity [eLife recent issues] 2013-04-02 08:16
We do not know how or why multicellularity evolved. We used the budding yeast, Saccharomyces cerevisiae, to ask whether nutrients that must be digested extracellularly select for the evolution of undifferentiated multicellularity. Because yeast use invertase to hydrolyze sucrose extracellularly and import the resulting monosaccharides, single cells cannot grow at low cell and sucrose concentrations. Three engineered strategies overcame this problem: forming multicellular clumps, importing sucrose before hydrolysis, and increasing invertase expression. We evolved populations in low sucrose to ask which strategy they would adopt. Of 12 successful clones, 11 formed multicellular clumps through incomplete cell separation, 10 increased invertase expression, none imported sucrose, and 11 increased hexose transporter expression, a strategy we had not engineered. Identifying causal mutations revealed genes and pathways, which frequently contributed to the evolved phenotype. Our study shows that combining rational design with experimental evolution can help evaluate hypotheses about evolutionary strategies.
Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization [eLife recent issues] 2013-04-02 08:16
A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment.
Mechanism for priming DNA synthesis by yeast DNA Polymerase {alpha} [eLife recent issues] 2013-04-02 08:16
The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment.
Basic research at the epicenter of an epidemic [eLife recent issues] 2013-04-02 08:16
William R Bishai, director of the KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH), argues that the best place to carry out research into a disease is in its midst.
A sticky solution [eLife recent issues] 2013-04-02 08:16
Selection favours single-celled mutants that stick together when a sugar needed for growth is in short supply, suggesting that multicellular life may have evolved as a by-product of selection for more efficient usage of resources.
Sugar promotes vegetative phase change in Arabidopsis thaliana by repressing the expression of MIR156A and MIR156C [eLife recent issues] 2013-03-26 09:31
Nutrients shape the growth, maturation, and aging of plants and animals. In plants, the juvenile to adult transition (vegetative phase change) is initiated by a decrease in miR156. In Arabidopsis, we found that exogenous sugar decreased the abundance of miR156, whereas reduced photosynthesis increased the level of this miRNA. This effect was correlated with a change in the timing of vegetative phase change, and was primarily attributable to a change in the expression of two genes, MIR156A and MIR156C, which were found to play dominant roles in this transition. The glucose-induced repression of miR156 was dependent on the signaling activity of HEXOKINASE1. We also show that the defoliation-induced increase in miR156 levels can be suppressed by exogenous glucose. These results provide a molecular link between nutrient availability and developmental timing in plants, and suggest that sugar is a component of the leaf signal that mediates vegetative phase change.
Sugar is an endogenous cue for juvenile-to-adult phase transition in plants [eLife recent issues] 2013-03-26 09:31
The transition from the juvenile to adult phase in plants is controlled by diverse exogenous and endogenous cues such as age, day length, light, nutrients, and temperature. Previous studies have shown that the gradual decline in microRNA156 (miR156) with age promotes the expression of adult traits. However, how age temporally regulates the abundance of miR156 is poorly understood. We show here that the expression of miR156 responds to sugar. Sugar represses miR156 expression at both the transcriptional level and post-transcriptional level through the degradation of miR156 primary transcripts. Defoliation and photosynthetic mutant assays further demonstrate that sugar from the pre-existing leaves acts as a mobile signal to repress miR156, and subsequently triggers the juvenile-to-adult phase transition in young leaf primordia. We propose that the gradual increase in sugar after seed germination serves as an endogenous cue for developmental timing in plants.
Sugars speed up the circle of life [eLife recent issues] 2013-03-26 09:31
By regulating the expression of key microRNA molecules, sugar levels in leaves control the transition from the juvenile to the adult form in plants.
The writing on the wall [eLife recent issues] 2013-03-26 09:31
The biomedical research enterprise in the US has become unsustainable and urgent action is needed to address a variety of problems, including a lack of innovation, an over-reliance on soft money for faculty salaries, the use of graduate students as a source of cheap labour, and a ‘holding tank’ full of talented postdocs with limited career opportunities.
Shedding new light on the origins of olfactory neurons [eLife recent issues] 2013-03-26 09:31
Sensory neurons in the nose of the zebrafish are derived from both neural crest cells and placode cells.
Selection of distinct populations of dentate granule cells in response to inputs as a mechanism for pattern separation in mice [eLife recent issues] 2013-03-20 06:48
The hippocampus is critical for episodic memory and computational studies have predicted specific functions for each hippocampal subregion. Particularly, the dentate gyrus (DG) is hypothesized to perform pattern separation by forming distinct representations of similar inputs. How pattern separation is achieved by the DG remains largely unclear. By examining neuronal activities at a population level, we revealed that, unlike CA1 neuron populations, dentate granule cell (DGC) ensembles activated by learning were not preferentially reactivated by memory recall. Moreover, when mice encountered an environment to which they had not been previously exposed, a novel DGC population—rather than the previously activated DGC ensembles that responded to past events—was selected to represent the new environmental inputs. This selection of a novel responsive DGC population could be triggered by small changes in environmental inputs. Therefore, selecting distinct DGC populations to represent similar but not identical inputs is a mechanism for pattern separation.
What's new is older [eLife recent issues] 2013-03-20 06:48
Distinct populations of active cells in the dentate gyrus of the hippocampus may facilitate the unique encoding of changes in the environment.
Sox10-dependent neural crest origin of olfactory microvillous neurons in zebrafish [eLife recent issues] 2013-03-19 09:01
The sense of smell in vertebrates is detected by specialized sensory neurons derived from the peripheral nervous system. Classically, it has been presumed that the olfactory placode forms all olfactory sensory neurons. In contrast, we show that the cranial neural crest is the primary source of microvillous sensory neurons within the olfactory epithelium of zebrafish embryos. Using photoconversion-based fate mapping and live cell tracking coupled with laser ablation, we followed neural crest precursors as they migrated from the neural tube to the nasal cavity. A subset that coexpressed Sox10 protein and a neurogenin1 reporter ingressed into the olfactory epithelium and differentiated into microvillous sensory neurons. Timed loss-of-function analysis revealed a critical role for Sox10 in microvillous neurogenesis. Taken together, these findings directly demonstrate a heretofore unknown contribution of the cranial neural crest to olfactory sensory neurons in zebrafish and provide important insights into the assembly of the nascent olfactory system.
Plants regenerated from tissue culture contain stable epigenome changes in rice [eLife recent issues] 2013-03-19 09:01
Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.
LIN-12/Notch signaling instructs postsynaptic muscle arm development by regulating UNC-40/DCC and MADD-2 in Caenorhabditis elegans [eLife recent issues] 2013-03-19 09:01
The diverse cell types and the precise synaptic connectivity between them are the cardinal features of the nervous system. Little is known about how cell fate diversification is linked to synaptic target choices. Here we investigate how presynaptic neurons select one type of muscles, vm2, as a synaptic target and form synapses on its dendritic spine-like muscle arms. We found that the Notch-Delta pathway was required to distinguish target from non-target muscles. APX-1/Delta acts in surrounding cells including the non-target vm1 to activate LIN-12/Notch in the target vm2. LIN-12 functions cell-autonomously to up-regulate the expression of UNC-40/DCC and MADD-2 in vm2, which in turn function together to promote muscle arm formation and guidance. Ectopic expression of UNC-40/DCC in non-target vm1 muscle is sufficient to induce muscle arm extension from these cells. Therefore, the LIN-12/Notch signaling specifies target selection by selectively up-regulating guidance molecules and forming muscle arms in target cells.
How to draw the line in biomedical research [eLife recent issues] 2013-03-19 09:01
The use of the least squares method to calculate the best-fitting line through a two-dimensional scatter plot typically requires the user to assume that one of the variables depends on the other. However, in many cases the relationship between the two variables is more complex, and it is not valid to say that one variable is independent and the other is dependent. When analysing such data researchers should consider plotting the three regression lines that can be calculated for any two-dimensional scatter plot.
Multitasking on the run [eLife recent issues] 2013-03-19 09:01
Researchers combine genetics and imaging to reveal that individual granule cells in the cerebellum integrate sensory and motor information.
Hip, hip, hooray! [eLife recent issues] 2013-03-12 03:05
X-rays are best known for showing where bones have fractured, but researchers can also use X-rays to investigate why bones break, which could lead to treatments that reduce the number of elderly people who suffer broken hips.
Building a super elongation complex for HIV [eLife recent issues] 2013-03-08 03:05
A better understanding of the host cell protein complex that helps HIV replicate inside cells offers the possibility of new therapeutic targets.
Kinesin-1 regulates dendrite microtubule polarity in Caenorhabditis elegans [eLife recent issues] 2013-03-06 06:45
In neurons, microtubules (MTs) span the length of both axons and dendrites, and the molecular motors use these intracellular ‘highways' to transport diverse cargo to the appropriate subcellular locations. Whereas axonal MTs are organized such that the plus-end is oriented out from the cell body, dendrites exhibit a mixed MTs polarity containing both minus-end-out and plus-end-out MTs. The molecular mechanisms underlying this differential organization, as well as its functional significance, are unknown. Here, we show that kinesin-1 is critical in establishing the characteristic minus-end-out MT organization of the dendrite in vivo. In unc-116 (kinesin-1/kinesin heavy chain) mutants, the dendritic MTs adopt an axonal-like plus-end-out organization. Kinesin-1 protein is able to cross-link anti-paralleled MTs in vitro. We propose that kinesin-1 regulates the dendrite MT polarity through directly gliding the plus-end-out MTs out of the dendrite using both the motor domain and the C-terminal MT-binding domain.
AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis [eLife recent issues] 2013-03-05 08:50
The clathrin adaptor complex AP2 is thought to be an obligate heterotetramer. We identify null mutations in the α subunit of AP2 in the nematode Caenorhabditis elegans. α-adaptin mutants are viable and the remaining μ2/β hemicomplex retains some function. Conversely, in μ2 mutants, the alpha/sigma2 hemicomplex is localized and is partially functional. α-μ2 double mutants disrupt both halves of the complex and are lethal. The lethality can be rescued by expression of AP2 components in the skin, which allowed us to evaluate the requirement for AP2 subunits at synapses. Mutations in either α or μ2 subunits alone reduce the number of synaptic vesicles by about 30%; however, simultaneous loss of both α and μ2 subunits leads to a 70% reduction in synaptic vesicles and the presence of large vacuoles. These data suggest that AP2 may function as two partially independent hemicomplexes.
De novo modeling of the F420-reducing [NiFe]-hydrogenase from a methanogenic archaeon by cryo-electron microscopy [eLife recent issues] 2013-03-05 08:50
Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure.
The AFF4 scaffold binds human P-TEFb adjacent to HIV Tat [eLife recent issues] 2013-03-05 08:50
Human positive transcription elongation factor b (P-TEFb) phosphorylates RNA polymerase II and regulatory proteins to trigger elongation of many gene transcripts. The HIV-1 Tat protein selectively recruits P-TEFb as part of a super elongation complex (SEC) organized on a flexible AFF1 or AFF4 scaffold. To understand this specificity and determine if scaffold binding alters P-TEFb conformation, we determined the structure of a tripartite complex containing the recognition regions of P-TEFb and AFF4. AFF4 meanders over the surface of the P-TEFb cyclin T1 (CycT1) subunit but makes no stable contacts with the CDK9 kinase subunit. Interface mutations reduced CycT1 binding and AFF4-dependent transcription. AFF4 is positioned to make unexpected direct contacts with HIV Tat, and Tat enhances P-TEFb affinity for AFF4. These studies define the mechanism of scaffold recognition by P-TEFb and reveal an unanticipated intersubunit pocket on the AFF4 SEC that potentially represents a target for therapeutic intervention against HIV/AIDS.
Mutual inhibition among postmitotic neurons regulates robustness of brain wiring in Drosophila [eLife recent issues] 2013-03-05 08:50
Brain connectivity maps display a delicate balance between individual variation and stereotypy, suggesting the existence of dedicated mechanisms that simultaneously permit and limit individual variation. We show that during the development of the Drosophila central nervous system, mutual inhibition among groups of neighboring postmitotic neurons during development regulates the robustness of axon target choice in a nondeterministic neuronal circuit. Specifically, neighboring postmitotic neurons communicate through Notch signaling during axonal targeting, to ensure balanced alternative axon target choices without a corresponding change in cell fate. Loss of Notch in postmitotic neurons modulates an axon's target choice. However, because neighboring axons respond by choosing the complementary target, the stereotyped connectivity pattern is preserved. In contrast, loss of Notch in clones of neighboring postmitotic neurons results in erroneous coinnervation by multiple axons. Our observations establish mutual inhibition of axonal target choice as a robustness mechanism for brain wiring and unveil a novel cell fate independent function for canonical Notch signaling.
What does it take to recruit and retain senior women faculty? [eLife recent issues] 2013-03-05 08:50
eLife deputy editor Fiona M Watt recounts some of her personal experiences as a senior female academic in a male-dominated environment.
Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates [eLife recent issues] 2013-02-26 08:01
Two-thirds of gene promoters in mammals are associated with regions of non-methylated DNA, called CpG islands (CGIs), which counteract the repressive effects of DNA methylation on chromatin. In cold-blooded vertebrates, computational CGI predictions often reside away from gene promoters, suggesting a major divergence in gene promoter architecture across vertebrates. By experimentally identifying non-methylated DNA in the genomes of seven diverse vertebrates, we instead reveal that non-methylated islands (NMIs) of DNA are a central feature of vertebrate gene promoters. Furthermore, NMIs are present at orthologous genes across vast evolutionary distances, revealing a surprising level of conservation in this epigenetic feature. By profiling NMIs in different tissues and developmental stages we uncover a unifying set of features that are central to the function of NMIs in vertebrates. Together these findings demonstrate an ancient logic for NMI usage at gene promoters and reveal an unprecedented level of epigenetic conservation across vertebrate evolution.
Convergence of pontine and proprioceptive streams onto multimodal cerebellar granule cells [eLife recent issues] 2013-02-26 08:01
Cerebellar granule cells constitute the majority of neurons in the brain and are the primary conveyors of sensory and motor-related mossy fiber information to Purkinje cells. The functional capability of the cerebellum hinges on whether individual granule cells receive mossy fiber inputs from multiple precerebellar nuclei or are instead unimodal; this distinction is unresolved. Using cell-type-specific projection mapping with synaptic resolution, we observed the convergence of separate sensory (upper body proprioceptive) and basilar pontine pathways onto individual granule cells and mapped this convergence across cerebellar cortex. These findings inform the long-standing debate about the multimodality of mammalian granule cells and substantiate their associative capacity predicted in the Marr-Albus theory of cerebellar function. We also provide evidence that the convergent basilar pontine pathways carry corollary discharges from upper body motor cortical areas. Such merging of related corollary and sensory streams is a critical component of circuit models of predictive motor control.
Bidding the CpG island goodbye [eLife recent issues] 2013-02-26 08:01
Experiments on seven vertebrates suggest that identifying the locations of islands of non-methylated DNA provides more insights into evolutionarily-conserved epigenetic regulatory elements than studies of CpG islands.
Correction: RecA filament sliding on DNA facilitates homology search [eLife recent issues] 2013-02-20 10:03
Mitotic spindle scaling during Xenopus development by kif2a and importin {alpha} [eLife recent issues] 2013-02-19 10:25
Early development of many animals is characterized by rapid cleavages that dramatically decrease cell size, but how the mitotic spindle adapts to changing cell dimensions is not understood. To identify mechanisms that scale the spindle during Xenopus laevis embryogenesis, we established an in vitro system using cytoplasmic extracts prepared from embryos that recapitulates in vivo spindle size differences between stage 3 (4 cells, 37 µm) and stage 8 (~4000 cells, 18 µm). We identified the kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor importin α, and activated in stage 8 when importin α partitions to a membrane pool. Altering spindle size in developing embryos impaired spindle orientation during metaphase, but chromosome segregation remained robust. Thus, spindle size in Xenopus development is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization.
UNC93B1 mediates differential trafficking of endosomal TLRs [eLife recent issues] 2013-02-19 10:25
UNC93B1, a multipass transmembrane protein required for TLR3, TLR7, TLR9, TLR11, TLR12, and TLR13 function, controls trafficking of TLRs from the endoplasmic reticulum (ER) to endolysosomes. The mechanisms by which UNC93B1 mediates these regulatory effects remain unclear. Here, we demonstrate that UNC93B1 enters the secretory pathway and directly controls the packaging of TLRs into COPII vesicles that bud from the ER. Unlike other COPII loading factors, UNC93B1 remains associated with the TLRs through post-Golgi sorting steps. Unexpectedly, these steps are different among endosomal TLRs. TLR9 requires UNC93B1-mediated recruitment of adaptor protein complex 2 (AP-2) for delivery to endolysosomes while TLR7, TLR11, TLR12, and TLR13 utilize alternative trafficking pathways. Thus, our study describes a mechanism for differential sorting of endosomal TLRs by UNC93B1, which may explain the distinct roles played by these receptors in certain autoimmune diseases.
Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates [eLife recent issues] 2013-02-19 10:25
Influenza virus penetrates cells by fusion of viral and endosomal membranes catalyzed by the viral hemagglutinin (HA). Structures of the initial and final states of the HA trimer define the fusion endpoints, but do not specify intermediates. We have characterized these transitions by analyzing low-pH-induced fusion kinetics of individual virions and validated the analysis by computer simulation. We detect initial engagement with the target membrane of fusion peptides from independently triggered HAs within the larger virus-target contact patch; fusion then requires engagement of three or four neighboring HA trimers. Effects of mutations in HA indicate that withdrawal of the fusion peptide from a pocket in the pre-fusion trimer is rate-limiting for both events, but the requirement for cooperative action of several HAs to bring the fusing membranes together leads to a long-lived intermediate state for single, extended HA trimers. This intermediate is thus a fundamental aspect of the fusion mechanism.
Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles [eLife recent issues] 2013-02-19 10:25
Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.
Developing cell biology [eLife recent issues] 2013-02-19 10:25
Experiments in Xenopus embryo extracts reveal that changes in cellular biochemistry cause mitotic spindles to decrease in size over the course of early development.
Different routes to the same destination [eLife recent issues] 2013-02-19 10:25
The toll-like receptors that detect viral DNA and viral RNA in cells take different paths from the endoplasmic reticulum to the endosome.
Direct detection pays off for electron cryo-microscopy [eLife recent issues] 2013-02-19 10:25
Improved electron detectors and image-processing techniques will allow the structures of macromolecules to be determined from tens of thousands of single-particle cryo-EM images, rather than the hundreds of thousands needed previously.
Correction: Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster [eLife recent issues] 2013-02-12 06:40
Dendritic cells loaded with FK506 kill T cells in an antigen-specific manner and prevent autoimmunity in vivo [eLife recent issues] 2013-02-05 12:35
FK506 (Tacrolimus) is a potent inhibitor of calcineurin that blocks IL2 production and is widely used to prevent transplant rejection and treat autoimmunity. FK506 treatment of dendritic cells (FKDC) limits their capacity to stimulate T cell responses. FK506 does not prevent DC survival, maturation, or costimulatory molecule expression, suggesting that the limited capacity of FKDC to stimulate T cells may be due to inhibition of calcineurin signaling in the DC. Instead, we demonstrate that DC inhibit T cells by sequestering FK506 and continuously releasing the drug over several days. T cells encountering FKDC proliferate but fail to upregulate the survival factor bcl-xl and die, and IL2 restores both bcl-xl and survival. In mice, FKDC act in an antigen-specific manner to inhibit T-cell mediated autoimmune arthritis. This establishes that DCs can act as a cellular drug delivery system to target antigen specific T cells.
Time line of redox events in aging postmitotic cells [eLife recent issues] 2013-02-05 12:35
The precise roles that oxidants play in lifespan and aging are still unknown. Here, we report the discovery that chronologically aging yeast cells undergo a sudden redox collapse, which affects over 80% of identified thiol-containing proteins. We present evidence that this redox collapse is not triggered by an increase in endogenous oxidants as would have been postulated by the free radical theory of aging. Instead it appears to be instigated by a substantial drop in cellular NADPH, which normally provides the electron source for maintaining cellular redox homeostasis. This decrease in NADPH levels occurs very early during lifespan and sets into motion a cascade that is predicted to down-regulate most cellular processes. Caloric restriction, a near-universal lifespan extending measure, increases NADPH levels and delays each facet of the cascade. Our studies reveal a time line of events leading up to the system-wide oxidation of the proteome days before cell death.
Hierarchical organization of context in the hippocampal episodic code [eLife recent issues] 2013-02-05 12:35
The hippocampal system appears to be critically important in establishing episodic memory of both internal and external events within contexts as well as spatial memory, which enables flexible spatial navigation. However, the neuronal substrates that function across different memories in the hippocampal system are poorly understood. I monitored large-scale activity patterns of hippocampal neuronal ensembles in rats performing a novel, continuous task that combined one visually guided and two memory-guided types of navigations in a constant environment. I found that the activity patterns of the hippocampal ensemble represent spatiotemporal contexts (journeys) constructed by temporally ordered past, present and expected future places in tandem with visually or mnemonically guided non-spatial contexts (task-demands) to form episodes. This finding therefore suggests that the hierarchical organization of contexts based on pattern separation and completion enables the hippocampus to play a dual role in spatial navigation and recall of episodic memory.
Recognition of familiar food activates feeding via an endocrine serotonin signal in Caenorhabditis elegans [eLife recent issues] 2013-02-05 12:35
Familiarity discrimination has a significant impact on the pattern of food intake across species. However, the mechanism by which the recognition memory controls feeding is unclear. Here, we show that the nematode Caenorhabditis elegans forms a memory of particular foods after experience and displays behavioral plasticity, increasing the feeding response when they subsequently recognize the familiar food. We found that recognition of familiar food activates the pair of ADF chemosensory neurons, which subsequently increase serotonin release. The released serotonin activates the feeding response mainly by acting humorally and directly activates SER-7, a type 7 serotonin receptor, in MC motor neurons in the feeding organ. Our data suggest that worms sense the taste and/or smell of novel bacteria, which overrides the stimulatory effect of familiar bacteria on feeding by suppressing the activity of ADF or its upstream neurons. Our study provides insight into the mechanism by which familiarity discrimination alters behavior.
A new answer to old questions [eLife recent issues] 2013-02-05 12:35
Sudden changes in the level of a coenzyme called NADPH might be the underlying cause of aging in cells.
RNA-programmed genome editing in human cells [eLife recent issues] 2013-01-29 08:01
Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.
Bacteria herald a new era of gene editing [eLife recent issues] 2013-01-29 08:01
The demonstration that nucleases guided by bacterial RNA can disrupt human genes represents a landmark in the rapidly developing field of genome engineering.
Changing the responses of cortical neurons from sub- to suprathreshold using single spikes in vivo [eLife recent issues] 2013-01-22 17:55
Action Potential (APs) patterns of sensory cortex neurons encode a variety of stimulus features, but how can a neuron change the feature to which it responds? Here, we show that in vivo a spike-timing-dependent plasticity (STDP) protocol—consisting of pairing a postsynaptic AP with visually driven presynaptic inputs—modifies a neurons' AP-response in a bidirectional way that depends on the relative AP-timing during pairing. Whereas postsynaptic APs repeatedly following presynaptic activation can convert subthreshold into suprathreshold responses, APs repeatedly preceding presynaptic activation reduce AP responses to visual stimulation. These changes were paralleled by restructuring of the neurons response to surround stimulus locations and membrane-potential time-course. Computational simulations could reproduce the observed subthreshold voltage changes only when presynaptic temporal jitter was included. Together this shows that STDP rules can modify output patterns of sensory neurons and the timing of single-APs plays a crucial role in sensory coding and plasticity.
NOVA-dependent regulation of cryptic NMD exons controls synaptic protein levels after seizure [eLife recent issues] 2013-01-22 17:55
The neuronal RNA binding protein NOVA regulates splicing, shuttles to the cytoplasm, and co-localizes with target transcripts in dendrites, suggesting links between splicing and local translation. Here we identified >200 transcripts showing NOVA-dependent changes in abundance, but, surprisingly, HITS-CLIP revealed NOVA binds these RNAs in introns rather than 3' UTRs. This led us to discover NOVA-regulated splicing of cryptic exons within these introns. These exons triggered nonsense mediated decay (NMD), as UPF1 and protein synthesis were required for NOVA's effect on RNA levels. Their regulation was dynamic and physiologically relevant. The NMD exons were regulated by seizures, which also induced changes in Nova subcellular localization and mediated large changes in synaptic proteins, including proteins implicated in familial epilepsy. Moreover, Nova haploinsufficient mice had spontaneous epilepsy. The data reveal a hidden means of dynamic RNA regulation linking electrical activity to splicing and protein output, and of mediating homeostatic excitation/inhibition balance in neurons.
A virus responds instantly to the presence of the vector on the host and forms transmission morphs [eLife recent issues] 2013-01-22 17:55
Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle.
Strong inter-population cooperation leads to partner intermixing in microbial communities [eLife recent issues] 2013-01-22 17:55
Patterns of spatial positioning of individuals within microbial communities are often critical to community function. However, understanding patterning in natural communities is hampered by the multitude of cell–cell and cell–environment interactions as well as environmental variability. Here, through simulations and experiments on communities in defined environments, we examined how ecological interactions between two distinct partners impacted community patterning. We found that in strong cooperation with spatially localized large fitness benefits to both partners, a unique pattern is generated: partners spatially intermixed by appearing successively on top of each other, insensitive to initial conditions and interaction dynamics. Intermixing was experimentally observed in two obligatory cooperative systems: an engineered yeast community cooperating through metabolite-exchanges and a methane-producing community cooperating through redox-coupling. Even in simulated communities consisting of several species, most of the strongly-cooperating pairs appeared intermixed. Thus, when ecological interactions are the major patterning force, strong cooperation leads to partner intermixing.
Cdc48/p97 promotes degradation of aberrant nascent polypeptides bound to the ribosome [eLife recent issues] 2013-01-22 17:55
Ubiquitin-dependent proteolysis can initiate at ribosomes for myriad reasons including misfolding of a nascent chain or stalling of the ribosome during translation of mRNA. Clearance of a stalled complex is required to recycle the ribosome for future use. Here we show that the ubiquitin (Ub) pathway segregase Cdc48/p97 and its adaptors Ufd1-Npl4 participate in ribosome-associated degradation (RAD) by mediating the clearance of ubiquitinated, tRNA-linked nascent peptides from ribosomes. Through characterization of both endogenously-generated and heterologous model substrates for the RAD pathway, we conclude that budding yeast Cdc48 functions downstream of the Ub ligases Ltn1 and Ubr1 to release nascent proteins from the ribosome so that they can be degraded by the proteasome. Defective RAD could contribute to the pathophysiology of human diseases caused by mutations in p97.
'Cryptic' exons reveal some of their secrets [eLife recent issues] 2013-01-22 17:55
By regulating the inclusion of ‘cryptic’ exons in messenger RNA in nerve cells, NOVA proteins are able to influence the abundance of the corresponding proteins.
Crossing oceans [eLife recent issues] 2013-01-22 17:55
Eve Marder explains why all scientists should spend time living and working in a foreign country.
How to train a neuron [eLife recent issues] 2013-01-22 17:55
A cellular learning rule known as spike-timing-dependent plasticity can form, reshape and erase the response preferences of visual cortex neurons.
Myosin motors fragment and compact membrane-bound actin filaments [eLife recent issues] 2013-01-08 08:50
Cell cortex remodeling during cell division is a result of myofilament-driven contractility of the cortical membrane-bound actin meshwork. Little is known about the interaction between individual myofilaments and membrane-bound actin filaments. Here we reconstituted a minimal actin cortex to directly visualize the action of individual myofilaments on membrane-bound actin filaments using TIRF microscopy. We show that synthetic myofilaments fragment and compact membrane-bound actin while processively moving along actin filaments. We propose a mechanism by which tension builds up between the ends of myofilaments, resulting in compressive stress exerted to single actin filaments, causing their buckling and breakage. Modeling of this mechanism revealed that sufficient force (~20 pN) can be generated by single myofilaments to buckle and break actin filaments. This mechanism of filament fragmentation and compaction may contribute to actin turnover and cortex reorganization during cytokinesis.
A novel GTP-binding protein-adaptor protein complex responsible for export of Vangl2 from the trans Golgi network [eLife recent issues] 2013-01-08 08:50
Planar cell polarity (PCP) requires the asymmetric sorting of distinct signaling receptors to distal and proximal surfaces of polarized epithelial cells. We have examined the transport of one PCP signaling protein, Vangl2, from the trans Golgi network (TGN) in mammalian cells. Using siRNA knockdown experiments, we find that the GTP-binding protein, Arfrp1, and the clathrin adaptor complex 1 (AP-1) are required for Vangl2 transport from the TGN. In contrast, TGN export of Frizzled 6, which localizes to the opposing epithelial surface from Vangl2, does not depend on Arfrp1 or AP-1. Mutagenesis studies identified a YYXXF sorting signal in the C-terminal cytosolic domain of Vangl2 that is required for Vangl2 traffic and interaction with the μ subunit of AP-1. We propose that Arfrp1 exposes a binding site on AP-1 that recognizes the Vangl2 sorting motif for capture into a transport vesicle destined for the proximal surface of a polarized epithelial cell.
Dual functions of TAF7L in adipocyte differentiation [eLife recent issues] 2013-01-08 08:50
The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPAR at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPAR. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPAR and promoters as a component of the core transcriptional machinery.
Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster [eLife recent issues] 2013-01-08 08:50
Coordinated walking in vertebrates and multi-legged invertebrates such as Drosophila melanogaster requires a complex neural network coupled to sensory feedback. An understanding of this network will benefit from systems such as Drosophila that have the ability to genetically manipulate neural activities. However, the fly's small size makes it challenging to analyze walking in this system. In order to overcome this limitation, we developed an optical method coupled with high-speed imaging that allows the tracking and quantification of gait parameters in freely walking flies with high temporal and spatial resolution. Using this method, we present a comprehensive description of many locomotion parameters, such as gait, tarsal positioning, and intersegmental and left-right coordination for wild type fruit flies. Surprisingly, we find that inactivation of sensory neurons in the fly's legs, to block proprioceptive feedback, led to deficient step precision, but interleg coordination and the ability to execute a tripod gait were unaffected.
Fruit flies step out [eLife recent issues] 2013-01-08 08:50
A method that can analyse the movements of Drosophila as they walk is a valuable addition to the tools available to neurobiologists, and has already led to insights into the interplay of central networks and sensory feedback in this model organism.
Faculty appointments and the record of scholarship [eLife recent issues] 2013-01-08 08:50
Academic review committees would benefit from more details about the contributions made by individual researchers to papers with multiple authors, and also from more information about other types of scholarly communication.
A novel role for lipid droplets in the organismal antibacterial response [eLife recent issues] 2012-01-01 03:00
We previously discovered histones bound to cytosolic lipid droplets (LDs); here we show that this forms a cellular antibacterial defense system. Sequestered on droplets under normal conditions, in the presence of bacterial lipopolysaccharide (LPS) or lipoteichoic acid (LTA), histones are released from the droplets and kill bacteria efficiently in vitro. Droplet-bound histones also function in vivo: when injected into Drosophila embryos lacking droplet-bound histones, bacteria grow rapidly. In contrast, bacteria injected into embryos with droplet-bound histones die. Embryos with droplet-bound histones displayed more than a fourfold survival advantage when challenged with four different bacterial species. Our data suggests that this intracellular antibacterial defense system may function in adult flies, and also potentially in mice.
Molecular architecture of human polycomb repressive complex 2 [eLife recent issues] 2012-01-01 03:00
Polycomb Repressive Complex 2 (PRC2) is essential for gene silencing, establishing transcriptional repression of specific genes by tri-methylating Lysine 27 of histone H3, a process mediated by cofactors such as AEBP2. In spite of its biological importance, little is known about PRC2 architecture and subunit organization. Here, we present the first three-dimensional electron microscopy structure of the human PRC2 complex bound to its cofactor AEBP2. Using a novel internal protein tagging-method, in combination with isotopic chemical cross-linking and mass spectrometry, we have localized all the PRC2 subunits and their functional domains and generated a detailed map of interactions. The position and stabilization effect of AEBP2 suggests an allosteric role of this cofactor in regulating gene silencing. Regions in PRC2 that interact with modified histone tails are localized near the methyltransferase site, suggesting a molecular mechanism for the chromatin-based regulation of PRC2 activity.
Herbivory-induced volatiles function as defenses increasing fitness of the native plant Nicotiana attenuata in nature [eLife recent issues] 2012-01-01 03:00
From an herbivore's first bite, plants release herbivory-induced plant volatiles (HIPVs) which can attract enemies of herbivores. However, other animals and competing plants can intercept HIPVs for their own use, and it remains unclear whether HIPVs serve as an indirect defense by increasing fitness for the emitting plant. In a 2-year field study, HIPV-emitting N. attenuata plants produced twice as many buds and flowers as HIPV-silenced plants, but only when native Geocoris spp. predators reduced herbivore loads (by 50%) on HIPV-emitters. In concert with HIPVs, plants also employ antidigestive trypsin protease inhibitors (TPIs), but TPI-producing plants were not fitter than TPI-silenced plants. TPIs weakened a specialist herbivore's behavioral evasive responses to simulated Geocoris spp. attack, indicating that TPIs function against specialists by enhancing indirect defense.
Nascent-Seq reveals novel features of mouse circadian transcriptional regulation [eLife recent issues] 2012-01-01 03:00
A substantial fraction of the metazoan transcriptome undergoes circadian oscillations in many cells and tissues. Based on the transcription feedback loops important for circadian timekeeping, it is commonly assumed that this mRNA cycling reflects widespread transcriptional regulation. To address this issue, we directly measured the circadian dynamics of mouse liver transcription using Nascent-Seq (genome-wide sequencing of nascent RNA). Although many genes are rhythmically transcribed, many rhythmic mRNAs manifest poor transcriptional rhythms, indicating a prominent contribution of post-transcriptional regulation to circadian mRNA expression. This analysis of rhythmic transcription also showed that the rhythmic DNA binding profile of the transcription factors CLOCK and BMAL1 does not determine the transcriptional phase of most target genes. This likely reflects gene-specific collaborations of CLK:BMAL1 with other transcription factors. These insights from Nascent-Seq indicate that it should have broad applicability to many other gene expression regulatory issues.
A bacterial sulfonolipid triggers multicellular development in the closest living relatives of animals [eLife recent issues] 2012-01-01 03:00
Bacterially-produced small molecules exert profound influences on animal health, morphogenesis, and evolution through poorly understood mechanisms. In one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta, we find that rosette colony development is induced by the prey bacterium Algoriphagus machipongonensis and its close relatives in the Bacteroidetes phylum. Here we show that a rosette inducing factor (RIF-1) produced by A. machipongonensis belongs to the small class of sulfonolipids, obscure relatives of the better known sphingolipids that play important roles in signal transmission in plants, animals, and fungi. RIF-1 has extraordinary potency (femtomolar, or 10–15 M) and S. rosetta can respond to it over a broad dynamic range—nine orders of magnitude. This study provides a prototypical example of bacterial sulfonolipids triggering eukaryotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals.
Foggy perception slows us down [eLife recent issues] 2012-01-01 03:00
Visual speed is believed to be underestimated at low contrast, which has been proposed as an explanation of excessive driving speed in fog. Combining psychophysics measurements and driving simulation, we confirm that speed is underestimated when contrast is reduced uniformly for all objects of the visual scene independently of their distance from the viewer. However, we show that when contrast is reduced more for distant objects, as is the case in real fog, visual speed is actually overestimated, prompting drivers to decelerate. Using an artificial anti-fog—that is, fog characterized by better visibility for distant than for close objects, we demonstrate for the first time that perceived speed depends on the spatial distribution of contrast over the visual scene rather than the global level of contrast per se. Our results cast new light on how reduced visibility conditions affect perceived speed, providing important insight into the human visual system.
DNA-PK is a DNA sensor for IRF-3-dependent innate immunity [eLife recent issues] 2012-01-01 03:00
Innate immunity is the first immunological defence against pathogens. During virus infection detection of nucleic acids is crucial for the inflammatory response. Here we identify DNA-dependent protein kinase (DNA-PK) as a DNA sensor that activates innate immunity. We show that DNA-PK acts as a pattern recognition receptor, binding cytoplasmic DNA and triggering the transcription of type I interferon (IFN), cytokine and chemokine genes in a manner dependent on IFN regulatory factor 3 (IRF-3), TANK-binding kinase 1 (TBK1) and stimulator of interferon genes (STING). Both cells and mice lacking DNA-PKcs show attenuated cytokine responses to both DNA and DNA viruses but not to RNA or RNA virus infection. DNA-PK has well-established functions in the DNA repair and V(D)J recombination, hence loss of DNA-PK leads to severe combined immunodeficiency (SCID). However, we now define a novel anti-microbial function for DNA-PK, a finding with implications for host defence, vaccine development and autoimmunity.
The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis [eLife recent issues] 2012-01-01 03:00
The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks both a Hac1/XBP1 ortholog and a UPR-dependent-transcriptional-program. Instead, Ire1 initiates the selective decay of a subset of ER-localized-mRNAs that is required to survive ER stress. We identified Bip1 mRNA, encoding a major ER-chaperone, as the sole mRNA cleaved upon Ire1 activation that escapes decay. Instead, truncation of its 3' UTR, including loss of its polyA tail, stabilized Bip1 mRNA, resulting in increased Bip1 translation. Thus, S. pombe uses a universally conserved stress-sensing machinery in novel ways to maintain homeostasis in the ER.
Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus [eLife recent issues] 2012-01-01 03:00
Human hepatitis B virus (HBV) infection and HBV-related diseases remain a major public health problem. Individuals coinfected with its satellite hepatitis D virus (HDV) have more severe disease. Cellular entry of both viruses is mediated by HBV envelope proteins. The pre-S1 domain of the large envelope protein is a key determinant for receptor(s) binding. However, the identity of the receptor(s) is unknown. Here, by using near zero distance photo-cross-linking and tandem affinity purification, we revealed that the receptor-binding region of pre-S1 specifically interacts with sodium taurocholate cotransporting polypeptide (NTCP), a multiple transmembrane transporter predominantly expressed in the liver. Silencing NTCP inhibited HBV and HDV infection, while exogenous NTCP expression rendered nonsusceptible hepatocarcinoma cells susceptible to these viral infections. Moreover, replacing amino acids 157–165 of nonfunctional monkey NTCP with the human counterpart conferred its ability in supporting both viral infections. Our results demonstrate that NTCP is a functional receptor for HBV and HDV.
Global divergence in critical income for adult and childhood survival: analyses of mortality using Michaelis-Menten [eLife recent issues] 2012-01-01 03:00
Life expectancy has risen sharply in the last 50 years. We applied the classic Michaelis–Menten enzyme kinetics to demonstrate a novel mathematical relationship of income to childhood (aged 0–5 years) and adult (aged 15–60 years) survival. We treat income as a substrate that is catalyzed to increase survival (from technologies that income buys) for 180 countries from 1970 and 2007. Michaelis–Menten kinetics permit estimates of maximal survival and, uniquely, the critical income needed to achieve half of the period-specific maximum. Maximum child and adult survival rose by about 1% per year. Critical incomes fell by half for children, but doubled for men. HIV infection and smoking account for some, but not all, of the rising critical incomes for adult survival. Altering the future cost curve for adult survival will require more widespread use of current interventions, most notably tobacco control, but also research to identify practicable low-cost drugs, diagnostics, and strategies.
The starvation hormone, fibroblast growth factor-21, extends lifespan in mice [eLife recent issues] 2012-01-01 03:00
Fibroblast growth factor-21 (FGF21) is a hormone secreted by the liver during fasting that elicits diverse aspects of the adaptive starvation response. Among its effects, FGF21 induces hepatic fatty acid oxidation and ketogenesis, increases insulin sensitivity, blocks somatic growth and causes bone loss. Here we show that transgenic overexpression of FGF21 markedly extends lifespan in mice without reducing food intake or affecting markers of NAD+ metabolism or AMP kinase and mTOR signaling. Transcriptomic analysis suggests that FGF21 acts primarily by blunting the growth hormone/insulin-like growth factor-1 signaling pathway in liver. These findings raise the possibility that FGF21 can be used to extend lifespan in other species.
RecA filament sliding on DNA facilitates homology search [eLife recent issues] 2012-01-01 03:00
During homologous recombination, RecA forms a helical filament on a single stranded (ss) DNA that searches for a homologous double stranded (ds) DNA and catalyzes the exchange of complementary base pairs to form a new heteroduplex. Using single molecule fluorescence imaging tools with high spatiotemporal resolution we characterized the encounter complex between the RecA filament and dsDNA. We present evidence in support of the ‘sliding model’ wherein a RecA filament diffuses along a dsDNA track. We further show that homology can be detected during sliding. Sliding occurs with a diffusion coefficient of approximately 8000 bp2/s allowing the filament to sample several hundred base pairs before dissociation. Modeling suggests that sliding can accelerate homology search by as much as 200 fold. Homology recognition can occur for as few as 6 nt of complementary basepairs with the recognition efficiency increasing for higher complementarity. Our data represents the first example of a DNA bound multi-protein complex which can slide along another DNA to facilitate target search.
Non-canonical TAF complexes regulate active promoters in human embryonic stem cells [eLife recent issues] 2012-01-01 03:00
The general transcription factor TFIID comprises the TATA-box-binding protein (TBP) and approximately 14 TBP-associated factors (TAFs). Here we find, unexpectedly, that undifferentiated human embryonic stem cells (hESCs) contain only six TAFs (TAFs 2, 3, 5, 6, 7 and 11), whereas following differentiation all TAFs are expressed. Directed and global chromatin immunoprecipitation analyses reveal an unprecedented promoter occupancy pattern: most active genes are bound by only TAFs 3 and 5 along with TBP, whereas the remaining active genes are bound by TBP and all six hESC TAFs. Consistent with these results, hESCs contain a previously undescribed complex comprising TAFs 2, 6, 7, 11 and TBP. Altering the composition of hESC TAFs, either by depleting TAFs that are present or ectopically expressing TAFs that are absent, results in misregulated expression of pluripotency genes and induction of differentiation. Thus, the selective expression and use of TAFs underlies the ability of hESCs to self-renew.
The activity-dependent histone variant H2BE modulates the life span of olfactory neurons [eLife recent issues] 2012-01-01 03:00
We have identified a replication-independent histone variant, Hist2h2be (referred to herein as H2be), which is expressed exclusively by olfactory chemosensory neurons. Levels of H2BE are heterogeneous among olfactory neurons, but stereotyped according to the identity of the co-expressed olfactory receptor (OR). Gain- and loss-of-function experiments demonstrate that changes in H2be expression affect olfactory function and OR representation in the adult olfactory epithelium. We show that H2BE expression is reduced by sensory activity and that it promotes neuronal cell death, such that inactive olfactory neurons display higher levels of the variant and shorter life spans. Post-translational modifications (PTMs) of H2BE differ from those of the canonical H2B, consistent with a role for H2BE in altering transcription. We propose a physiological function for H2be in modulating olfactory neuron population dynamics to adapt the OR repertoire to the environment.
Chromatin is an ancient innovation conserved between Archaea and Eukarya [eLife recent issues] 2012-01-01 03:00
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ~147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved –1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.
Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1 [eLife recent issues] 2012-01-01 03:00
Doxorubicin is used extensively for chemotherapy of diverse types of cancer, yet the mechanism through which it inhibits proliferation of cancer cells remains unclear. Here we report that doxorubicin stimulates de novo synthesis of ceramide, which in turn activates CREB3L1, a transcription factor synthesized as a membrane-bound precursor. Doxorubicin stimulates proteolytic cleavage of CREB3L1 by Site-1 Protease and Site-2 Protease, allowing the NH2-terminal domain of CREB3L1 to enter the nucleus where it activates transcription of genes encoding inhibitors of the cell cycle, including p21. Knockdown of CREB3L1 mRNA in human hepatoma Huh7 cells and immortalized human fibroblast SV589 cells conferred increased resistance to doxorubicin, whereas overexpression of CREB3L1 in human breast cancer MCF-7 cells markedly enhanced the sensitivity of these cells to doxorubicin. These results suggest that measurement of CREB3L1 expression may be a useful biomarker in identifying cancer cells sensitive to doxorubicin.
Population structuring of multi-copy, antigen-encoding genes in Plasmodium falciparum [eLife recent issues] 2012-01-01 03:00
The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier ‘strain theory’ by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity.
Sequence specific detection of bacterial 23S ribosomal RNA by TLR13 [eLife recent issues] 2012-01-01 03:00
Toll-like receptors (TLRs) detect microbial infections and trigger innate immune responses. Among vertebrate TLRs, the role of TLR13 and its ligand are unknown. Here we show that TLR13 detects the 23S ribosomal RNA of both gram-positive and gram-negative bacteria. A sequence containing 13 nucleotides near the active site of 23S rRNA ribozyme, which catalyzes peptide bond synthesis, was both necessary and sufficient to trigger TLR13-dependent interleukin-1β production. Single point mutations within this sequence destroyed the ability of the 23S rRNA to stimulate the TLR13 pathway. Knockout of TLR13 in mice abolished the induction of interleukin-1β and other cytokines by the 23S rRNA sequence. Thus, TLR13 detects bacterial RNA with exquisite sequence specificity.
Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion [eLife recent issues] 2012-01-01 03:00
The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways.
Meiosis I chromosome segregation is established through regulation of microtubule-kinetochore interactions [eLife recent issues] 2012-01-01 03:00
During meiosis, a single round of DNA replication is followed by two consecutive rounds of nuclear divisions called meiosis I and meiosis II. In meiosis I, homologous chromosomes segregate, while sister chromatids remain together. Determining how this unusual chromosome segregation behavior is established is central to understanding germ cell development. Here we show that preventing microtubule–kinetochore interactions during premeiotic S phase and prophase I is essential for establishing the meiosis I chromosome segregation pattern. Premature interactions of kinetochores with microtubules transform meiosis I into a mitosis-like division by disrupting two key meiosis I events: coorientation of sister kinetochores and protection of centromeric cohesin removal from chromosomes. Furthermore we find that restricting outer kinetochore assembly contributes to preventing premature engagement of microtubules with kinetochores. We propose that inhibition of microtubule–kinetochore interactions during premeiotic S phase and prophase I is central to establishing the unique meiosis I chromosome segregation pattern.
Elba, a novel developmentally regulated chromatin boundary factor is a hetero-tripartite DNA binding complex [eLife recent issues] 2012-01-01 03:00
Chromatin boundaries subdivide eukaryotic chromosomes into functionally autonomous domains of genetic activity. This subdivision insulates genes and/or regulatory elements within a domain from promiscuous interactions with nearby domains. While it was previously assumed that the chromosomal domain landscape is fixed, there is now growing evidence that the landscape may be subject to tissue and stage specific regulation. Here we report the isolation and characterization of a novel developmentally restricted boundary factor, Elba. We show that Elba is an unusual hetero-tripartite protein complex that requires all three proteins for DNA binding and insulator activity.
Membrane immersion allows rhomboid proteases to achieve specificity by reading transmembrane segment dynamics [eLife recent issues] 2012-01-01 03:00
Rhomboid proteases reside within cellular membranes, but the advantage of this unusual environment is unclear. We discovered membrane immersion allows substrates to be identified in a fundamentally-different way, based initially upon exposing ‘masked’ conformational dynamics of transmembrane segments rather than sequence-specific binding. EPR and CD spectroscopy revealed that the membrane restrains rhomboid gate and substrate conformation to limit proteolysis. True substrates evolved intrinsically-unstable transmembrane helices that both become unstructured when not supported by the membrane, and facilitate partitioning into the hydrophilic, active-site environment. Accordingly, manipulating substrate and gate dynamics in living cells shifted cleavage sites in a manner incompatible with extended sequence binding, but correlated with a membrane-and-helix-exit propensity scale. Moreover, cleavage of diverse non-substrates was provoked by single-residue changes that destabilize transmembrane helices. Membrane immersion thus bestows rhomboid proteases with the ability to identify substrates primarily based on reading their intrinsic transmembrane dynamics.
Morphologic diversity of cutaneous sensory afferents revealed by genetically directed sparse labeling [eLife recent issues] 2012-01-01 03:00
The diversity of cutaneous sensory afferents has been studied by many investigators using behavioral, physiologic, molecular, and genetic approaches. Largely missing, thus far, is an analysis of the complete morphologies of individual afferent arbors. Here we present a survey of cutaneous sensory arbor morphologies in hairy skin of the mouse using genetically-directed sparse labeling with a sensory neuron-specific alkaline phosphatase reporter. Quantitative analyses of 719 arbors, among which 77 were fully reconstructed, reveal 10 morphologically distinct types. Among the two types with the largest arbors, one contacts ~200 hair follicles with circumferential endings and a second is characterized by a densely ramifying arbor with one to several thousand branches and a total axon length between one-half and one meter. These observations constrain models of receptive field size and structure among cutaneous sensory neurons, and they raise intriguing questions regarding the cellular and developmental mechanisms responsible for this morphological diversity.
Distinct gating mechanisms revealed by the structures of a multi-ligand gated K+ channel [eLife recent issues] 2012-01-01 03:00
The gating ring-forming RCK domain regulates channel gating in response to various cellular chemical stimuli in eukaryotic Slo channel families and the majority of ligand-gated prokaryotic K+ channels and transporters. Here we present structural and functional studies of a dual RCK-containing, multi-ligand gated K+ channel from Geobacter sulfurreducens, named GsuK. We demonstrate that ADP and NAD+ activate the GsuK channel, whereas Ca2+ serves as an allosteric inhibitor. Multiple crystal structures elucidate the structural basis of multi-ligand gating in GsuK, and also reveal a unique ion conduction pore with segmented inner helices. Structural comparison leads us to propose a novel pore opening mechanics that is distinct from other K+ channels.
KDM2B links the Polycomb Repressive Complex 1 (PRC1) to recognition of CpG islands [eLife recent issues] 2012-01-01 03:00
CpG islands (CGIs) are associated with most mammalian gene promoters. A subset of CGIs act as polycomb response elements (PREs) and are recognized by the polycomb silencing systems to regulate expression of genes involved in early development. How CGIs function mechanistically as nucleation sites for polycomb repressive complexes remains unknown. Here we discover that KDM2B (FBXL10) specifically recognizes non-methylated DNA in CGIs and recruits the polycomb repressive complex 1 (PRC1). This contributes to histone H2A lysine 119 ubiquitylation (H2AK119ub1) and gene repression. Unexpectedly, we also find that CGIs are occupied by low levels of PRC1 throughout the genome, suggesting that the KDM2B-PRC1 complex may sample CGI-associated genes for susceptibility to polycomb-mediated silencing. These observations demonstrate an unexpected and direct link between recognition of CGIs by KDM2B and targeting of the polycomb repressive system. This provides the basis for a new model describing the functionality of CGIs as mammalian PREs.
Indirect routes to reproductive success [eLife recent issues] 2012-01-01 03:00
By comparing wild-type and transgenic tobacco plants in a natural ecosystem, researchers have confirmed that the indirect defence mechanisms employed by plants to fend off herbivorous insects can increase Darwinian fitness.
Molecular clue links bacteria to the origin of animals [eLife recent issues] 2012-01-01 03:00
Bacteria have a role in the formation of colonies by a species of single-celled organisms whose ancestors gave rise to the animals, which suggests that bacteria might also have influenced the origin of multicellularity in animals.
New twists in the unfolded protein response [eLife recent issues] 2012-01-01 03:00
The response of S. pombe, also known as fission yeast, to misfolded proteins involves mechanisms that have not been observed in other species.
rsEGFP2 enables fast RESOLFT nanoscopy of living cells [eLife recent issues] 2012-01-01 03:00
The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25–250 times faster recordings.
Launching eLife, Part 1 [eLife recent issues] 2012-01-01 03:00
The new open-access journal eLife has launched, making its first content available in PubMed Central. In addition to publishing science of the highest quality, the journal aims to improve both the peer-review process and the presentation of new research results.
New ideas on how drivers perceive speed emerge from the fog [eLife recent issues] 2012-01-01 03:00
Experiments with a driving simulator contradict previous results by showing that car drivers slow down in fog. However, other forms of reduced visibility can cause drivers to speed up.
Could a hormone point the way to life extension? [eLife recent issues] 2012-01-01 03:00
Mice that have been genetically modified to produce high levels of fibroblast growth factor-21 live longer than mice with normal levels of this hormone.
Getting to grips with hepatitis [eLife recent issues] 2012-01-01 03:00
The receptor that allows hepatitis B and hepatitis D viruses to enter human liver cells has been identified as a protein that transports bile acids in the liver.
Histones join the fight against bacteria inside cells [eLife recent issues] 2012-01-01 03:00
Experiments on Drosophila have shown that the histones that are normally bound to lipid droplets inside cells can be released to provide protection against infection.
Modelling dynamics in protein crystal structures by ensemble refinement [eLife recent issues] 2012-01-01 03:00
Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships.
How keeping active pays off in the olfactory system [eLife recent issues] 2012-01-01 03:00
A protein that is found in the main olfactory epithelium of mice ensures that odour-sensing neurons that are active to have longer lifespans than those that are inactive.
Enzymes provide demographers with food for thought [eLife recent issues] 2012-01-01 03:00
Life expectancy has increased by 20 years since the middle of the last century, but children under five have fared better than adult males.
Sliding to the rescue of damaged DNA [eLife recent issues] 2012-01-01 03:00
Single-molecule imaging experiments have shed new light on the methods used by the enzyme RecA to align single- and double-stranded DNA so that double-strand breaks can be repaired.
Bad medicine [eLife recent issues] 2012-01-01 03:00
In his new book Ben Goldacre argues that the pharmaceutical industry is in poor health and in urgent need of treatment. Richard Smith agrees.
Nerve endings reveal hidden diversity in the skin [eLife recent issues] 2012-01-01 03:00
Experiments on transgenic mice have revealed that the morphologies of sensory neurons in the skin of mice are more complex and diverse than expected.
A good life [eLife recent issues] 2012-01-01 03:00
Following a career in science involves long hours and hard work, but as Eve Marder explains in the first of a series of columns, it can also be extremely rewarding.
Launching eLife, Part 2 [eLife recent issues] 2012-01-01 03:00
With a commitment to open access and innovation in peer review, eLife aims to publish important results in the life and biomedical sciences in a flexible digital format that allows authors to present their work in full, including the key data on which the conclusions are based.
Mathematics and malaria [eLife recent issues] 2012-01-01 03:00
Populations of malaria parasites are strongly influenced by their interactions with human immunity, and a better understanding of these interactions can help to explain why the risks from this potentially lethal disease vary according to location and age.
Helping chromosomes and chromatids stay on track [eLife recent issues] 2012-01-01 03:00
The prevention of premature interactions between microtubules and kinetochores is essential to ensuring that meiosis produces gametes with the correct number of chromosomes.
How does doxorubicin work? [eLife recent issues] 2012-01-01 03:00
A new mechanism involving cleavage of a transcription factor called CREB3L1 has been proposed to explain the anti-tumour effects of doxorubicin.
Nanoscopy at low light intensities shows its potential [eLife recent issues] 2012-01-01 03:00
A new form of green fluorescent protein allows super-resolution imaging to be performed faster on living cells with low radiation doses.
All together now [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
All together now
Nature 502, 7473 (2013). doi:10.1038/502593a
Proposals to bring hydrofluorocarbons under the auspices of the Montreal Protocol provide a simple test of the international community’s commitment to tackling climate change.
Time to talk [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Time to talk
Nature 502, 7473 (2013). doi:10.1038/502593b
Online discussion is an essential aspect of the post-publication review of findings.
Playful paradoxes [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Playful paradoxes
Nature 502, 7473 (2013). doi:10.1038/502594a
A half-century of Doctor Who has shown the dramatic possibilities of science in the arts.
Science’s rightful place is in service of society [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Science’s rightful place is in service of society
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502595a
Author: Daniel Sarewitz
Science policy must concentrate less on how much money is spent, and more on how to translate investments into public good, says Daniel Sarewitz.
Ecology: Hunting leads to a leap in lizards [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Ecology: Hunting leads to a leap in lizards
Nature 502, 7473 (2013). doi:10.1038/502596a
Traditional hunting seems to boost lizard populations in Australia.Rebecca Bliege Bird at Stanford University, California, and her colleagues found that numbers of sand monitor lizards (Varanus gouldii) in the Western Desert were largest where there was most hunting — lizard burrows were
Remote Sensing: Illicit gold rush in Peruvian Amazon [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Remote Sensing: Illicit gold rush in Peruvian Amazon
Nature 502, 7473 (2013). doi:10.1038/502596b
Gold mines across the Peruvian Amazon increased by more than 400% from 1999 to 2012, and are now the main cause of deforestation there.A team led by Gregory Asner of the Carnegie Institution for Science in Stanford, California, validated the results from satellite data
Materials science: Water sculptures crafted in oil [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Materials science: Water sculptures crafted in oil
Nature 502, 7473 (2013). doi:10.1038/502596c
Water droplets suspended in oil can be shaped into ellipsoids, tubes or even fish-like forms (pictured). Thomas Russell at the University of Massachusetts in Amherst and his team added chemically modified polymers and polystyrene nanoparticles to water droplets in oil.These components attract
Cancer: Antibody creeps up on cancer [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Cancer: Antibody creeps up on cancer
Nature 502, 7473 (2013). doi:10.1038/502596d
An antibody that becomes active only when it encounters tumours could provide a path to safer cancer drugs.Although gentler than other cancer drugs, side effects from antibodies still limit the dosage that patients can receive. To reduce toxicity, Henry Lowman of CytomX, a biotechnology
Climate science: Meandering winds precede heat spells [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Climate science: Meandering winds precede heat spells
Nature 502, 7473 (2013). doi:10.1038/502597a
Although the deadly heatwaves that afflict the United States arise from chaotic forces, they could be predicted up to three weeks before they hit.As yet, heatwaves cannot be foreseen until about ten days in advance. However, in a 12,000-year computer simulation of general atmospheric
Planetary science: Why Martian craters are flat [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Planetary science: Why Martian craters are flat
Nature 502, 7473 (2013). doi:10.1038/502597b
When meteorites pummelled a young Mars, they may have obliterated enough of its crust to allow the planet's mantle to well up and trigger volcano-like activity across the red planet.A team led by Christopher Edwards of the California Institute of Technology in Pasadena looked
Neuroscience: Rats propelled by electric pulses [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Neuroscience: Rats propelled by electric pulses
Nature 502, 7473 (2013). doi:10.1038/502597c
Deep-brain stimulation can help rats with injured spinal cords to walk. After severing a large portion of neural fibres in rats' spines, researchers led by Lukas Bachmann at the University of Zurich in Switzerland applied electrical pulses to a brainstem region that controls locomotion.This
Evolution: Human ancestor had small thumbs [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Evolution: Human ancestor had small thumbs
Nature 502, 7473 (2013). doi:10.1038/502597d
Fossil analysis reveals that an ancestor of modern humans would have made a terrible hitch-hiker.Past reconstructions of the hands of the hominin Australopithecus afarensis assigned scattered bones to individuals and single fingers. Campbell Rolian at the University of Calgary, Canada, and Adam Gordon
Virology: Not all dormant HIV is defective [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Virology: Not all dormant HIV is defective
Nature 502, 7473 (2013). doi:10.1038/502597e
Viral 'sleeper agents' in human genomes could make HIV even harder to cure than expected.Dormant HIV sequences called proviruses embed themselves in the DNA of immune-system cells, thwarting efforts to eliminate HIV infection. However, in laboratory tests, less than 1% of proviruses could be
Alzheimer's disease: Tau tangles exposed [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Alzheimer's disease: Tau tangles exposed
Nature 502, 7473 (2013). doi:10.1038/502597f
Tangles of the tau protein are found in the brains of people who have died with Alzheimer's disease, but establishing techniques to see the protein in living people has been difficult.A team led by Makoto Higuchi at the National Institute of Radiological Sciences in
Seven days: 25–31 October 2013 [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Seven days: 25–31 October 2013
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502598a
The week in science: PubMed pilots online commenting, NASA laser communications set speed record, and Greenland lifts ban on uranium mining.
Polio risk looms over Europe [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Polio risk looms over Europe
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502601a
Author: Declan Butler
Cases in Syria highlight vulnerability of nearby countries to the viral disease.
Root of maths genius sought [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Root of maths genius sought
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502602a
Author: Erika Check Hayden
Entrepreneur’s ‘Project Einstein’ taps 400 top academics for their DNA.
Black holes shrink but endure [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Black holes shrink but endure
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502603a
Author: Ron Cowen
Theorist’s idea takes on information-preservation problem.
Lightning network tested out in Guinea [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Lightning network tested out in Guinea
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502604a
Author: Jeff Tollefson
Project to forecast African storms provides cheap alternative to radar-based weather services.
Astronomers revisit dwarf stars’ promise [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Astronomers revisit dwarf stars’ promise
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502606a
Author: Eugenie Samuel Reich
Kepler data spur searches for habitable planets around small, low-temperature stars.
Farmers dig into soil quality [Nature - Issue - nature.com science feeds] 2013-10-28 20:00
Farmers dig into soil quality
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502607a
Author: Quirin Schiermeier
Analytical technique promises to match fertilizers to soil in bid to boost yields in Africa.
Astronomy: Southern star [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Astronomy: Southern star
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502608a
Author: Linda Nordling
Can the Southern African Large Telescope live up to its potential?
Environment: Waste production must peak this century [Nature - Issue - nature.com science feeds]
Environment: Waste production must peak this century
Nature 502, 7473 (2013). doi:10.1038/502615a
Authors: Daniel Hoornweg, Perinaz Bhada-Tata & Chris Kennedy
Without drastic action, population growth and urbanization will outpace waste reduction, warn Daniel Hoornweg, Perinaz Bhada-Tata and Chris Kennedy.
Climate change: Melting glaciers bring energy uncertainty [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Climate change: Melting glaciers bring energy uncertainty
Nature 502, 7473 (2013). doi:10.1038/502617a
Author: Javaid Laghari
Countries should work together to understand how the Himalayan thaw will affect hydroelectric energy, says Javaid R. Laghari.
Physics: The time lord and fellow travellers [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Physics: The time lord and fellow travellers
Nature 502, 7473 (2013). doi:10.1038/502620a
Author: Andrew Jaffe
As television's time-bending Doctor Who turns 50, Andrew Jaffe explores time travel in fiction and science.
Books in brief [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Books in brief
Nature 502, 7473 (2013). doi:10.1038/502621a
Author: Barbara Kiser
The Manhattan Project's impact reverberated beyond the atomic bomb, reveals Angela Creager in this lucid scientific history. It paved the way for the Oak Ridge National Laboratory in Tennessee to mass-produce radioisotopes — elemental variants that emit radiation — for peacetime use. These newly abundant
Psychology: The appetite for right [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Psychology: The appetite for right
Nature 502, 7473 (2013). doi:10.1038/502622a
Author: John Whitfield
John Whitfield explores two studies that take us from infant ethics to moral choices faced by adults in society.
Space science: Zero-gravity hero [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Space science: Zero-gravity hero
Nature 502, 7473 (2013). doi:10.1038/502623a
Author: John Gilbey
John Gilbey is gripped by the memoir of Chris Hadfield, a former International Space Station commander.
Anthropocene: keep the guard up [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Anthropocene: keep the guard up
Nature 502, 7473 (2013). doi:10.1038/502624a
Author: Tim Caro
Chris Thomas writes that recent gains in species numbers associated with climate warming could more than balance species losses (Nature502, 7; 201310.1038/502007a). But conservation is not just about total species richness — it is also about functioning ecosystems.Unlike
Anthropocene: action makes sense [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Anthropocene: action makes sense
Nature 502, 7473 (2013). doi:10.1038/502624b
Authors: Daniel Simberloff & Piero Genovesi
Chris Thomas argues that natural hybridization of invasive and native species could have unforeseen ecological benefits (Nature502, 7; 201310.1038/502007a). However, it is dangerous to underestimate non-native species' potential for ecological and economic damage, which can manifest after decades of
IPCC: Climate panel is ripe for examination [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
IPCC: Climate panel is ripe for examination
Nature 502, 7473 (2013). doi:10.1038/502624c
Authors: Mike Hulme & Martin Mahony
Sociologists of science wish to study the Intergovernmental Panel on Climate Change (IPCC) for the same reason that they want to examine other loci at which scientific knowledge is made — whether in a laboratory, the field, a museum or at a conference. We too
City trees: Urban greening needs better data [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
City trees: Urban greening needs better data
Nature 502, 7473 (2013). doi:10.1038/502624d
Author: Diane E. Pataki
Current urban-greening programmes are all too often based on inadequate data (see, for example, C. T.Driscollet al. BioScience62, 354–366; 2012), and models for estimating the value of urban vegetation are largely untested. To make substantive progress
Visual acuity: Bird vision offers sharp insight [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Visual acuity: Bird vision offers sharp insight
Nature 502, 7473 (2013). doi:10.1038/502624e
Author: Damian Scarf
I find the explosion of interest in the visual system of mice surprising, given that murine eyesight is equivalent to 20/2,000 vision in humans. With their 20/50 vision, pigeons might offer a less “blurry picture” of human 20/20 visual acuity (Nature502, 156
David Hunter Hubel (1926–2013) [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
David Hunter Hubel (1926–2013)
Nature 502, 7473 (2013). doi:10.1038/502625a
Author: Carla J. Shatz
Neuroscientist who helped to reveal how the brain processes visual information.
Europe: Swedish success story [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Europe: Swedish success story
Nature 502, 7473 (2013). doi:10.1038/nj7473-711a
Author: Paul Smaglik
Institutions shake off rivalries to build scientific collaborations and hire world-class talent.
Turning point: Mark Matthews [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Turning point: Mark Matthews
Nature 502, 7473 (2013). doi:10.1038/nj7473-713a
Author: Virginia Gewin
Former English teacher pursues app development to help patients with mental illness.
Mobility disincentive [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Mobility disincentive
Nature 502, 7473 (2013). doi:10.1038/nj7473-713b
Switching institutions or research groups can delay tenure.
Inaccurate predictions [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Inaccurate predictions
Nature 502, 7473 (2013). doi:10.1038/nj7473-713c
Peer review fails at predicting success.
Real-time online advice [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Real-time online advice
Nature 502, 7473 (2013). doi:10.1038/nj7473-713d
Online portal coaches prospective postgraduates on study programmes in Europe.
Deep impressions [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Medical imaging [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Scans: Enhanced medical vision [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Scans: Enhanced medical vision
Nature. doi:10.1038/502S82a
Author: Brian Owens
The ability to look inside the human body without using a scalpel has revolutionized how we diagnose and treat illness and injury. By Brian Owens.
Alzheimer's disease: Mapping the brain's decline [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Alzheimer's disease: Mapping the brain's decline
Nature. doi:10.1038/502S84a
Author: Sarah C. P. Williams
Imaging the brains of Alzheimer's patients provides insights into the way this insidious disease progresses.
Inflammation: A complex problem [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Inflammation: A complex problem
Nature. doi:10.1038/502S86a
Author: Katharine Gammon
Multi-protein inflammasomes are being implicated in a surprising number of diseases, and researchers are keen to find out why.
Surgery: The eyes of the operation [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Surgery: The eyes of the operation
Nature. doi:10.1038/502S88a
Author: Jessica Wright
Real-time imaging of a patient's body is guiding surgeons and radiologists past healthy tissue to the diseased cells.
Technology: Multiple exposure [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Technology: Multiple exposure
Nature. doi:10.1038/502S90a
Author: Neil Savage
Combining imaging techniques can provide a wealth of information about disease.
Software: The computer will see you now [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Software: The computer will see you now
Nature. doi:10.1038/502S92a
Author: Katherine Bourzac
From image-analysis software to lens-free microscopes that fit on a mobile phone, new tools are providing pathologists with clearer and more informative images.
Perspective: The big picture [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Perspective: The big picture
Nature. doi:10.1038/502S95a
Author: Alan Moody
Many medical images are used once then filed away. This trove of clinical data should be made available to biomedical researchers, says Alan Moody.
Next-generation scans: Seeing into the future [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Next-generation scans: Seeing into the future
Nature. doi:10.1038/502S96a
Author: Peter Gwynne
From magnetically tagged sugar to smoke-sensing surgical knives and beams of high-energy protons, the next wave of imaging technologies will provide a clearer view of the body.
Correction [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Correction
Nature 502, 7473 (2013). http://www.nature.com/doifinder/10.1038/502607b
The News story ‘Final word is near on dark-matter signal’ (Nature502, 421; 2013), wrongly quoted Jonathan Feng as saying the LUX results could rule out all types of neutralino — he believes that only some types would be ruled out.
Ecology: Drivers of decoupling in drylands [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Ecology: Drivers of decoupling in drylands
Nature 502, 7473 (2013). doi:10.1038/502628a
Authors: David A. Wardle
A study reveals that increasing aridity alters the balance of carbon, nitrogen and phosphorus in dryland soils, providing insight into how global climate change will affect soil fertility and ecosystem services. See Letter p.672
Structural biology: Pivotal findings for a transcription machine [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Structural biology: Pivotal findings for a transcription machine
Nature 502, 7473 (2013). doi:10.1038/nature12700
Authors: Joost Zomerdijk
Crystal structures of the complete RNA polymerase I complex are now revealed. The structures link the opening and closing of this enzyme's DNA-binding cleft to the control of transcription. See Articles p.644 & p.650
Quantum physics: Single electrons pop out of the Fermi sea [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Quantum physics: Single electrons pop out of the Fermi sea
Nature 502, 7473 (2013). doi:10.1038/nature12699
Authors: Christian Flindt
The ability to control individual electrons in an electronic conductor would pave the way for novel quantum technologies. Single electrons emerging from a sea of their fellows in a nanoscale electrode can now be generated. See Letter p.659
50 & 100 Years Ago [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
50 & 100 Years Ago
Nature 502, 7473 (2013). doi:10.1038/502631a
50 Years AgoDuring the past year, reports of a remarkable case of 'digital vision' have percolated into Britain from the U.S.S.R. ... The subject ... whose personality is admittedly abnormal, is said to have trained herself to distinguish colours and forms by means of
Biophysics: Rough passage across a barrier [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Biophysics: Rough passage across a barrier
Nature 502, 7473 (2013). doi:10.1038/nature12697
Authors: Benjamin Schuler & Jane Clarke
The dynamics of chemical reactions in solution are described by Kramers' theory, but the parameters involved have eluded direct measurement. A study of protein folding reveals how this problem can be overcome. See Letter p.685
Water management: The data gap [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Water management: The data gap
Nature 502, 7473 (2013). doi:10.1038/502633a
Authors: Blanca Jiménez Cisneros
A comprehensive search identifies a global dearth of data on the generation, treatment and use of wastewater. Remedying this situation will help policy-makers to better legislate for the management of this precious resource.
Marine biology: Coral animals combat stress with sulphur [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Marine biology: Coral animals combat stress with sulphur
Nature 502, 7473 (2013). doi:10.1038/nature12698
Authors: Graham Jones
Photosynthetic algal symbionts of corals produce sulphur substances that are involved in the regulation of ocean temperatures. In a twist to the tale, it emerges that coral animals produce the same compounds. See Letter p.677
Arteriolar niches maintain haematopoietic stem cell quiescence [Nature - Issue - nature.com science feeds] 2013-10-08 20:00
Arteriolar niches maintain haematopoietic stem cell quiescence
Nature 502, 7473 (2013). doi:10.1038/nature12612
Authors: Yuya Kunisaki, Ingmar Bruns, Christoph Scheiermann, Jalal Ahmed, Sandra Pinho, Dachuan Zhang, Toshihide Mizoguchi, Qiaozhi Wei, Daniel Lucas, Keisuke Ito, Jessica C. Mar, Aviv Bergman & Paul S. Frenette
Cell cycle quiescence is a critical feature contributing to haematopoietic stem cell (HSC) maintenance. Although various candidate stromal cells have been identified as potential HSC niches, the spatial localization of quiescent HSCs in the bone marrow remains unclear. Here, using a novel approach that combines
Crystal structure of the 14-subunit RNA polymerase I [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Crystal structure of the 14-subunit RNA polymerase I
Nature 502, 7473 (2013). doi:10.1038/nature12636
Authors: Carlos Fernández-Tornero, María Moreno-Morcillo, Umar J. Rashid, Nicholas M. I. Taylor, Federico M. Ruiz, Tim Gruene, Pierre Legrand, Ulrich Steuerwald & Christoph W. Müller
Protein biosynthesis depends on the availability of ribosomes, which in turn relies on ribosomal RNA production. In eukaryotes, this process is carried out by RNA polymerase I (Pol I), a 14-subunit enzyme, the activity of which is a major determinant of cell growth. Here we
RNA polymerase I structure and transcription regulation [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
RNA polymerase I structure and transcription regulation
Nature 502, 7473 (2013). doi:10.1038/nature12712
Authors: Christoph Engel, Sarah Sainsbury, Alan C. Cheung, Dirk Kostrewa & Patrick Cramer
Transcription of ribosomal RNA by RNA polymerase (Pol) I initiates ribosome biogenesis and regulates eukaryotic cell growth. The crystal structure of Pol I from the yeast Saccharomyces cerevisiae at 2.8 Å resolution reveals all 14 subunits of the 590-kilodalton enzyme, and shows differences to Pol II.
A uniform metal distribution in the intergalactic medium of the Perseus cluster of galaxies [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
A uniform metal distribution in the intergalactic medium of the Perseus cluster of galaxies
Nature 502, 7473 (2013). doi:10.1038/nature12646
Authors: Norbert Werner, Ondrej Urban, Aurora Simionescu & Steven W. Allen
Most of the metals (elements heavier than helium) produced by stars in the member galaxies of clusters currently reside within the hot, X-ray-emitting intra-cluster gas. Observations of X-ray line emission from this intergalactic medium have suggested a relatively small cluster-to-cluster scatter outside the cluster centres and enrichment with iron out to large radii, leading to the idea that the metal enrichment occurred early in the history of the Universe. Models with early enrichment predict a uniform metal distribution at large radii in clusters, whereas those with late-time enrichment are expected to introduce significant spatial variations of the metallicity. To discriminate clearly between these competing models, it is essential to test for potential inhomogeneities by measuring the abundances out to large radii along multiple directions in clusters, which has not hitherto been done. Here we report a remarkably uniform iron abundance, as a function of radius and azimuth, that is statistically consistent with a constant value of ZFe = 0.306 ± 0.012 in solar units out to the edge of the nearby Perseus cluster. This homogeneous distribution requires that most of the metal enrichment of the intergalactic medium occurred before the cluster formed, probably more than ten billion years ago, during the period of maximal star formation and black hole activity.
Minimal-excitation states for electron quantum optics using levitons [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Minimal-excitation states for electron quantum optics using levitons
Nature 502, 7473 (2013). doi:10.1038/nature12713
Authors: J. Dubois, T. Jullien, F. Portier, P. Roche, A. Cavanna, Y. Jin, W. Wegscheider, P. Roulleau & D. C. Glattli
The on-demand generation of pure quantum excitations is important for the operation of quantum systems, but it is particularly difficult for a system of fermions. This is because any perturbation affects all states below the Fermi energy, resulting in a complex superposition of particle and hole excitations. However, it was predicted nearly 20 years ago that a Lorentzian time-dependent potential with quantized flux generates a minimal excitation with only one particle and no hole. Here we report that such quasiparticles (hereafter termed levitons) can be generated on demand in a conductor by applying voltage pulses to a contact. Partitioning the excitations with an electronic beam splitter generates a current noise that we use to measure their number. Minimal-excitation states are observed for Lorentzian pulses, whereas for other pulse shapes there are significant contributions from holes. Further identification of levitons is provided in the energy domain with shot-noise spectroscopy, and in the time domain with electronic Hong–Ou–Mandel noise correlations. The latter, obtained by colliding synchronized levitons on a beam splitter, exemplifies the potential use of levitons for quantum information: using linear electron quantum optics in ballistic conductors, it is possible to imagine flying-qubit operation in which the Fermi statistics are exploited to entangle synchronized electrons emitted by distinct sources. Compared with electron sources based on quantum dots, the generation of levitons does not require delicate nanolithography, considerably simplifying the circuitry for scalability. Levitons are not limited to carrying a single charge, and so in a broader context n-particle levitons could find application in the study of full electron counting statistics. But they can also carry a fraction of charge if they are implemented in Luttinger liquids or in fractional quantum Hall edge channels; this allows the study of Abelian and non-Abelian quasiparticles in the time domain. Finally, the generation technique could be applied to cold atomic gases, leading to the possibility of atomic levitons.
Coupling a single electron to a Bose–Einstein condensate [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Coupling a single electron to a Bose–Einstein condensate
Nature 502, 7473 (2013). doi:10.1038/nature12592
Authors: Jonathan B. Balewski, Alexander T. Krupp, Anita Gaj, David Peter, Hans Peter Büchler, Robert Löw, Sebastian Hofferberth & Tilman Pfau
The coupling of electrons to matter lies at the heart of our understanding of material properties such as electrical conductivity. Electron–phonon coupling can lead to the formation of a Cooper pair out of two repelling electrons, which forms the basis for Bardeen–Cooper–Schrieffer superconductivity. Here we study the interaction of a single localized electron with a Bose–Einstein condensate and show that the electron can excite phonons and eventually trigger a collective oscillation of the whole condensate. We find that the coupling is surprisingly strong compared to that of ionic impurities, owing to the more favourable mass ratio. The electron is held in place by a single charged ionic core, forming a Rydberg bound state. This Rydberg electron is described by a wavefunction extending to a size of up to eight micrometres, comparable to the dimensions of the condensate. In such a state, corresponding to a principal quantum number of n = 202, the Rydberg electron is interacting with several tens of thousands of condensed atoms contained within its orbit. We observe surprisingly long lifetimes and finite size effects caused by the electron exploring the outer regions of the condensate. We anticipate future experiments on electron orbital imaging, the investigation of phonon-mediated coupling of single electrons, and applications in quantum optics.
Gradual demise of a thin southern Laurentide ice sheet recorded by Mississippi drainage [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Gradual demise of a thin southern Laurentide ice sheet recorded by Mississippi drainage
Nature 502, 7473 (2013). doi:10.1038/nature12609
Authors: Andrew D. Wickert, Jerry X. Mitrovica, Carlie Williams & Robert S. Anderson
At the Last Glacial Maximum (LGM), about 21,000 years before present, land-based ice sheets held enough water to reduce global mean sea level by 130 metres. Yet after decades of study, major uncertainties remain as to the distribution of that ice. Here we test four reconstructions of North American deglacial ice-sheet history by quantitatively connecting them to high-resolution oxygen isotope (δ18O) records from the Gulf of Mexico using a water mixing model. For each reconstruction, we route meltwater and seasonal runoff through the time-evolving Mississippi drainage basin, which co-evolves with ice geometry and changing topography as ice loads deform the solid Earth and produce spatially variable sea level in a process known as glacial isostatic adjustment. The δ18O records show that the Mississippi-drained southern Laurentide ice sheet contributed only 5.4 ± 2.1 metres to global sea level rise, of which 0.66 ± 0.07 metres were released during the meltwater pulse 1A event 14,650–14,310 years before present, far less water than previously thought. In contrast, the three reconstructions based on glacial isostatic adjustment overpredict the δ18O-based post-LGM meltwater volume by a factor of 1.6 to 3.6. The fourth reconstruction, which is based on ice physics, has a low enough Mississippi-routed meltwater discharge to be consistent with δ18O constraints, but also contains the largest LGM North American ice volume. This suggests that modelling based on ice physics may be the best way of matching isotopic records while also sequestering enough water in the North American ice sheets to match the observed LGM sea level fall.
Decoupling of soil nutrient cycles as a function of aridity in global drylands [Nature - Issue - nature.com science feeds] 2013-10-29 20:00
Decoupling of soil nutrient cycles as a function of aridity in global drylands
Nature 502, 7473 (2013). doi:10.1038/nature12670
Authors: Manuel Delgado-Baquerizo, Fernando T. Maestre, Antonio Gallardo, Matthew A. Bowker, Matthew D. Wallenstein, Jose Luis Quero, Victoria Ochoa, Beatriz Gozalo, Miguel García-Gómez, Santiago Soliveres, Pablo García-Palacios, Miguel Berdugo, Enrique Valencia, Cristina Escolar, Tulio Arredondo, Claudia Barraza-Zepeda, Donaldo Bran, José Antonio Carreira, Mohamed Chaieb, Abel A. Conceição, Mchich Derak, David J. Eldridge, Adrián Escudero, Carlos I. Espinosa, Juan Gaitán, M. Gabriel Gatica, Susana Gómez-González, Elizabeth Guzman, Julio R. Gutiérrez, Adriana Florentino, Estela Hepper, Rosa M. Hernández, Elisabeth Huber-Sannwald, Mohammad Jankju, Jushan Liu, Rebecca L. Mau, Maria Miriti, Jorge Monerris, Kamal Naseri, Zouhaier Noumi, Vicente Polo, Aníbal Prina, Eduardo Pucheta, Elizabeth Ramírez, David A. Ramírez-Collantes, Roberto Romão, Matthew Tighe, Duilio Torres, Cristian Torres-Díaz, Eugene D. Ungar, James Val, Wanyoike Wamiti, Deli Wang & Eli Zaady
The biogeochemical cycles of carbon (C), nitrogen (N) and phosphorus (P) are interlinked by primary production, respiration and decomposition in terrestrial ecosystems. It has been suggested that the C, N and P cycles could become uncoupled under rapid climate change because of the different degrees of control exerted on the supply of these elements by biological and geochemical processes. Climatic controls on biogeochemical cycles are particularly relevant in arid, semi-arid and dry sub-humid ecosystems (drylands) because their biological activity is mainly driven by water availability. The increase in aridity predicted for the twenty-first century in many drylands worldwide may therefore threaten the balance between these cycles, differentially affecting the availability of essential nutrients. Here we evaluate how aridity affects the balance between C, N and P in soils collected from 224 dryland sites from all continents except Antarctica. We find a negative effect of aridity on the concentration of soil organic C and total N, but a positive effect on the concentration of inorganic P. Aridity is negatively related to plant cover, which may favour the dominance of physical processes such as rock weathering, a major source of P to ecosystems, over biological processes that provide more C and N, such as litter decomposition. Our findings suggest that any predicted increase in aridity with climate change will probably reduce the concentrations of N and C in global drylands, but increase that of P. These changes would uncouple the C, N and P cycles in drylands and could negatively affect the provision of key services provided by these ecosystems.
DMSP biosynthesis by an animal and its role in coral thermal stress response [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
DMSP biosynthesis by an animal and its role in coral thermal stress response
Nature 502, 7473 (2013). doi:10.1038/nature12677
Authors: Jean-Baptiste Raina, Dianne M. Tapiolas, Sylvain Forêt, Adrian Lutz, David Abrego, Janja Ceh, François O. Seneca, Peta L. Clode, David G. Bourne, Bette L. Willis & Cherie A. Motti
Globally, reef-building corals are the most prolific producers of dimethylsulphoniopropionate (DMSP), a central molecule in the marine sulphur cycle and precursor of the climate-active gas dimethylsulphide. At present, DMSP production by corals is attributed entirely to their algal endosymbiont, Symbiodinium. Combining chemical, genomic and molecular approaches, we show that coral juveniles produce DMSP in the absence of algal symbionts. DMSP levels increased up to 54% over time in newly settled coral juveniles lacking algal endosymbionts, and further increases, up to 76%, were recorded when juveniles were subjected to thermal stress. We uncovered coral orthologues of two algal genes recently identified in DMSP biosynthesis, strongly indicating that corals possess the enzymatic machinery necessary for DMSP production. Our results overturn the paradigm that photosynthetic organisms are the sole biological source of DMSP, and highlight the double jeopardy represented by worldwide declining coral cover, as the potential to alleviate thermal stress through coral-produced DMSP declines correspondingly.
Structural insight into magnetochrome-mediated magnetite biomineralization [Nature - Issue - nature.com science feeds] 2013-10-05 20:00
Structural insight into magnetochrome-mediated magnetite biomineralization
Nature 502, 7473 (2013). doi:10.1038/nature12573
Authors: Marina I. Siponen, Pierre Legrand, Marc Widdrat, Stephanie R. Jones, Wei-Jia Zhang, Michelle C. Y. Chang, Damien Faivre, Pascal Arnoux & David Pignol
Magnetotactic bacteria align along the Earth’s magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(ii)Fe(iii)2O4) or greigite (Fe(ii)Fe(iii)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(ii) and iron(iii) is clear, little is known about the biological mechanisms controlling their ratio. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(ii) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(iii) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.
Single-molecule fluorescence probes dynamics of barrier crossing [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Single-molecule fluorescence probes dynamics of barrier crossing
Nature 502, 7473 (2013). doi:10.1038/nature12649
Authors: Hoi Sung Chung & William A. Eaton
Kramers developed the theory on how chemical reaction rates are influenced by the viscosity of the medium. At the viscosity of water, the kinetics of unimolecular reactions are described by diffusion of a Brownian particle over a free-energy barrier separating reactants and products. For reactions in solution this famous theory extended Eyring’s transition state theory, and is widely applied in physics, chemistry and biology, including to reactions as complex as protein folding. Because the diffusion coefficient of Kramers’ theory is determined by the dynamics in the sparsely populated region of the barrier top, its properties have not been directly measured for any molecular system. Here we show that the Kramers diffusion coefficient and free-energy barrier can be characterized by measuring the temperature- and viscosity-dependence of the transition path time for protein folding. The transition path is the small fraction of an equilibrium trajectory for a single molecule when the free-energy barrier separating two states is actually crossed. Its duration, the transition path time, can now be determined from photon trajectories for single protein molecules undergoing folding/unfolding transitions. Our finding of a long transition path time with an unusually small solvent viscosity dependence suggests that internal friction as well as solvent friction determine the Kramers diffusion coefficient for α-helical proteins, as opposed to a breakdown of his theory, which occurs for many small-molecule reactions. It is noteworthy that the new and fundamental information concerning Kramers’ theory and the dynamics of barrier crossings obtained here come from experiments on a protein rather than a much simpler chemical or physical system.
Photosynthetic entrainment of the Arabidopsis thaliana circadian clock [Nature - Issue - nature.com science feeds] 2013-10-22 20:00
Photosynthetic entrainment of the Arabidopsis thaliana circadian clock
Nature 502, 7473 (2013). doi:10.1038/nature12603
Authors: Michael J. Haydon, Olga Mielczarek, Fiona C. Robertson, Katharine E. Hubbard & Alex A. R. Webb
Circadian clocks provide a competitive advantage in an environment that is heavily influenced by the rotation of the Earth, by driving daily rhythms in behaviour, physiology and metabolism in bacteria, fungi, plants and animals. Circadian clocks comprise transcription–translation feedback loops, which are entrained by environmental signals such as light and temperature to adjust the phase of rhythms to match the local environment. The production of sugars by photosynthesis is a key metabolic output of the circadian clock in plants. Here we show that these rhythmic, endogenous sugar signals can entrain circadian rhythms in Arabidopsis thaliana by regulating the gene expression of circadian clock components early in the photoperiod, thus defining a ‘metabolic dawn’. By inhibiting photosynthesis, we demonstrate that endogenous oscillations in sugar levels provide metabolic feedback to the circadian oscillator through the morning-expressed gene PSEUDO-RESPONSE REGULATOR 7 (PRR7), and we identify that prr7 mutants are insensitive to the effects of sucrose on the circadian period. Thus, photosynthesis has a marked effect on the entrainment and maintenance of robust circadian rhythms in A. thaliana, demonstrating that metabolism has a crucial role in regulation of the circadian clock.
Synthetic non-oxidative glycolysis enables complete carbon conservation [Nature - Issue - nature.com science feeds] 2013-09-28 20:00
Synthetic non-oxidative glycolysis enables complete carbon conservation
Nature 502, 7473 (2013). doi:10.1038/nature12575
Authors: Igor W. Bogorad, Tzu-Shyang Lin & James C. Liao
Glycolysis, or its variations, is a fundamental metabolic pathway in life that functions in almost all organisms to decompose external or intracellular sugars. The pathway involves the partial oxidation and splitting of sugars to pyruvate, which in turn is decarboxylated to produce acetyl-coenzyme A (CoA) for various biosynthetic purposes. The decarboxylation of pyruvate loses a carbon equivalent, and limits the theoretical carbon yield to only two moles of two-carbon (C2) metabolites per mole of hexose. This native route is a major source of carbon loss in biorefining and microbial carbon metabolism. Here we design and construct a non-oxidative, cyclic pathway that allows the production of stoichiometric amounts of C2 metabolites from hexose, pentose and triose phosphates without carbon loss. We tested this pathway, termed non-oxidative glycolysis (NOG), in vitro and in vivo in Escherichia coli. NOG enables complete carbon conservation in sugar catabolism to acetyl-CoA, and can be used in conjunction with CO2 fixation and other one-carbon (C1) assimilation pathways to achieve a 100% carbon yield to desirable fuels and chemicals.
Discovery of new enzymes and metabolic pathways by using structure and genome context [Nature - Issue - nature.com science feeds] 2013-09-21 20:00
Discovery of new enzymes and metabolic pathways by using structure and genome context
Nature 502, 7473 (2013). doi:10.1038/nature12576
Authors: Suwen Zhao, Ritesh Kumar, Ayano Sakai, Matthew W. Vetting, B. McKay Wood, Shoshana Brown, Jeffery B. Bonanno, Brandan S. Hillerich, Ronald D. Seidel, Patricia C. Babbitt, Steven C. Almo, Jonathan V. Sweedler, John A. Gerlt, John E. Cronan & Matthew P. Jacobson
Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computation-guided strategies for functional discovery with ‘metabolite docking’ to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by ‘genome neighbourhoods’ (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by ‘predicting’ the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-l-proline betaine (tHyp-B) and cis-4-hydroxy-d-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.
Meiotic chromosome structures constrain and respond to designation of crossover sites [Nature - Issue - nature.com science feeds] 2013-10-08 20:00
Meiotic chromosome structures constrain and respond to designation of crossover sites
Nature 502, 7473 (2013). doi:10.1038/nature12577
Authors: Diana E. Libuda, Satoru Uzawa, Barbara J. Meyer & Anne M. Villeneuve
Crossover recombination events between homologous chromosomes are required to form chiasmata, temporary connections between homologues that ensure their proper segregation at meiosis I. Despite this requirement for crossovers and an excess of the double-strand DNA breaks that are the initiating events for meiotic recombination, most organisms make very few crossovers per chromosome pair. Moreover, crossovers tend to inhibit the formation of other crossovers nearby on the same chromosome pair, a poorly understood phenomenon known as crossover interference. Here we show that the synaptonemal complex, a meiosis-specific structure that assembles between aligned homologous chromosomes, both constrains and is altered by crossover recombination events. Using a cytological marker of crossover sites in Caenorhabditis elegans, we show that partial depletion of the synaptonemal complex central region proteins attenuates crossover interference, increasing crossovers and reducing the effective distance over which interference operates, indicating that synaptonemal complex proteins limit crossovers. Moreover, we show that crossovers are associated with a local 0.4–0.5-micrometre increase in chromosome axis length. We propose that meiotic crossover regulation operates as a self-limiting system in which meiotic chromosome structures establish an environment that promotes crossover formation, which in turn alters chromosome structure to inhibit other crossovers at additional sites.
Visualizing virus assembly intermediates inside marine cyanobacteria [Nature - Issue - nature.com science feeds] 2013-10-08 20:00
Visualizing virus assembly intermediates inside marine cyanobacteria
Nature 502, 7473 (2013). doi:10.1038/nature12604
Authors: Wei Dai, Caroline Fu, Desislava Raytcheva, John Flanagan, Htet A. Khant, Xiangan Liu, Ryan H. Rochat, Cameron Haase-Pettingell, Jacqueline Piret, Steve J. Ludtke, Kuniaki Nagayama, Michael F. Schmid, Jonathan A. King & Wah Chiu
Cyanobacteria are photosynthetic organisms responsible for ∼25% of organic carbon fixation on the Earth. These bacteria began to convert solar energy and carbon dioxide into bioenergy and oxygen more than two billion years ago. Cyanophages, which infect these bacteria, have an important role in regulating the marine ecosystem by controlling cyanobacteria community organization and mediating lateral gene transfer. Here we visualize the maturation process of cyanophage Syn5 inside its host cell, Synechococcus, using Zernike phase contrast electron cryo-tomography (cryoET). This imaging modality yields dramatic enhancement of image contrast over conventional cryoET and thus facilitates the direct identification of subcellular components, including thylakoid membranes, carboxysomes and polyribosomes, as well as phages, inside the congested cytosol of the infected cell. By correlating the structural features and relative abundance of viral progeny within cells at different stages of infection, we identify distinct Syn5 assembly intermediates. Our results indicate that the procapsid releases scaffolding proteins and expands its volume at an early stage of genome packaging. Later in the assembly process, we detected full particles with a tail either with or without an additional horn. The morphogenetic pathway we describe here is highly conserved and was probably established long before that of double-stranded DNA viruses infecting more complex organisms.
An Evolution-Based Approach to De Novo Protein Design and Case Study on Mycobacterium tuberculosis [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Pralay Mitra, David Shultis, Jeffrey R. Brender, Jeff Czajka, David Marsh, Felicia Gray, Tomasz Cierpicki, Yang Zhang
Computational protein design is a reverse procedure of protein folding and structure prediction, where constructing structures from evolutionarily related proteins has been demonstrated to be the most reliable method for protein 3-dimensional structure prediction. Following this spirit, we developed a novel method to design new protein sequences based on evolutionarily related protein families. For a given target structure, a set of proteins having similar fold are identified from the PDB library by structural alignments. A structural profile is then constructed from the protein templates and used to guide the conformational search of amino acid sequence space, where physicochemical packing is accommodated by single-sequence based solvation, torsion angle, and secondary structure predictions. The method was tested on a computational folding experiment based on a large set of 87 protein structures covering different fold classes, which showed that the evolution-based design significantly enhances the foldability and biological functionality of the designed sequences compared to the traditional physics-based force field methods. Without using homologous proteins, the designed sequences can be folded with an average root-mean-square-deviation of 2.1 Å to the target. As a case study, the method is extended to redesign all 243 structurally resolved proteins in the pathogenic bacteria Mycobacterium tuberculosis, which is the second leading cause of death from infectious disease. On a smaller scale, five sequences were randomly selected from the design pool and subjected to experimental validation. The results showed that all the designed proteins are soluble with distinct secondary structure and three have well ordered tertiary structure, as demonstrated by circular dichroism and NMR spectroscopy. Together, these results demonstrate a new avenue in computational protein design that uses knowledge of evolutionary conservation from protein structural families to engineer new protein molecules of improved fold stability and biological functionality.Novel Methods for Analysing Bacterial Tracks Reveal Persistence in Rhodobacter sphaeroides [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Gabriel Rosser, Alexander G. Fletcher, David A. Wilkinson, Jennifer A. de Beyer, Christian A. Yates, Judith P. Armitage, Philip K. Maini, Ruth E. Baker
Tracking bacteria using video microscopy is a powerful experimental approach to probe their motile behaviour. The trajectories obtained contain much information relating to the complex patterns of bacterial motility. However, methods for the quantitative analysis of such data are limited. Most swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. It is therefore necessary to segment observed tracks into swimming and reorientation phases to extract useful statistics. We present novel robust analysis tools to discern these two phases in tracks. Our methods comprise a simple and effective protocol for removing spurious tracks from tracking datasets, followed by analysis based on a two-state hidden Markov model, taking advantage of the availability of mutant strains that exhibit swimming-only or reorientating-only motion to generate an empirical prior distribution. Using simulated tracks with varying levels of added noise, we validate our methods and compare them with an existing heuristic method. To our knowledge this is the first example of a systematic assessment of analysis methods in this field. The new methods are substantially more robust to noise and introduce less systematic bias than the heuristic method. We apply our methods to tracks obtained from the bacterial species Rhodobacter sphaeroides and Escherichia coli. Our results demonstrate that R. sphaeroides exhibits persistence over the course of a tumbling event, which is a novel result with important implications in the study of this and similar species.Exploring Protein-Peptide Binding Specificity through Computational Peptide Screening [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Arnab Bhattacherjee, Stefan Wallin
The binding of short disordered peptide stretches to globular protein domains is important for a wide range of cellular processes, including signal transduction, protein transport, and immune response. The often promiscuous nature of these interactions and the conformational flexibility of the peptide chain, sometimes even when bound, make the binding specificity of this type of protein interaction a challenge to understand. Here we develop and test a Monte Carlo-based procedure for calculating protein-peptide binding thermodynamics for many sequences in a single run. The method explores both peptide sequence and conformational space simultaneously by simulating a joint probability distribution which, in particular, makes searching through peptide sequence space computationally efficient. To test our method, we apply it to 3 different peptide-binding protein domains and test its ability to capture the experimentally determined specificity profiles. Insight into the molecular underpinnings of the observed specificities is obtained by analyzing the peptide conformational ensembles of a large number of binding-competent sequences. We also explore the possibility of using our method to discover new peptide-binding pockets on protein structures.Ten Simple Rules for Reproducible Computational Research [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Geir Kjetil Sandve, Anton Nekrutenko, James Taylor, Eivind Hovig
Comprehensive Repertoire of Foldable Regions within Whole Genomes [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Guilhem Faure, Isabelle Callebaut
In order to get a comprehensive repertoire of foldable domains within whole proteomes, including orphan domains, we developed a novel procedure, called SEG-HCA. From only the information of a single amino acid sequence, SEG-HCA automatically delineates segments possessing high densities in hydrophobic clusters, as defined by Hydrophobic Cluster Analysis (HCA). These hydrophobic clusters mainly correspond to regular secondary structures, which together form structured or foldable regions. Genome-wide analyses revealed that SEG-HCA is opposite of disorder predictors, both addressing distinct structural states. Interestingly, there is however an overlap between the two predictions, including small segments of disordered sequences, which undergo coupled folding and binding. SEG-HCA thus gives access to these specific domains, which are generally poorly represented in domain databases. Comparison of the whole set of SEG-HCA predictions with the Conserved Domain Database (CDD) also highlighted a wide proportion of predicted large (length >50 amino acids) segments, which are CDD orphan. These orphan sequences may either correspond to highly divergent members of already known families or belong to new families of domains. Their comprehensive description thus opens new avenues to investigate new functional and/or structural features, which remained so far uncovered. Altogether, the data described here provide new insights into the protein architecture and organization throughout the three kingdoms of life.Integrative Modelling of the Influence of MAPK Network on Cancer Cell Fate Decision [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Luca Grieco, Laurence Calzone, Isabelle Bernard-Pierrot, François Radvanyi, Brigitte Kahn-Perlès, Denis Thieffry
The Mitogen-Activated Protein Kinase (MAPK) network consists of tightly interconnected signalling pathways involved in diverse cellular processes, such as cell cycle, survival, apoptosis and differentiation. Although several studies reported the involvement of these signalling cascades in cancer deregulations, the precise mechanisms underlying their influence on the balance between cell proliferation and cell death (cell fate decision) in pathological circumstances remain elusive. Based on an extensive analysis of published data, we have built a comprehensive and generic reaction map for the MAPK signalling network, using CellDesigner software. In order to explore the MAPK responses to different stimuli and better understand their contributions to cell fate decision, we have considered the most crucial components and interactions and encoded them into a logical model, using the software GINsim. Our logical model analysis particularly focuses on urinary bladder cancer, where MAPK network deregulations have often been associated with specific phenotypes. To cope with the combinatorial explosion of the number of states, we have applied novel algorithms for model reduction and for the compression of state transition graphs, both implemented into the software GINsim. The results of systematic simulations for different signal combinations and network perturbations were found globally coherent with published data. In silico experiments further enabled us to delineate the roles of specific components, cross-talks and regulatory feedbacks in cell fate decision. Finally, tentative proliferative or anti-proliferative mechanisms can be connected with established bladder cancer deregulations, namely Epidermal Growth Factor Receptor (EGFR) over-expression and Fibroblast Growth Factor Receptor 3 (FGFR3) activating mutations.Natural, Persistent Oscillations in a Spatial Multi-Strain Disease System with Application to Dengue [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by José Lourenço, Mario Recker
Many infectious diseases are not maintained in a state of equilibrium but exhibit significant fluctuations in prevalence over time. For pathogens that consist of multiple antigenic types or strains, such as influenza, malaria or dengue, these fluctuations often take on the form of regular or irregular epidemic outbreaks in addition to oscillatory prevalence levels of the constituent strains. To explain the observed temporal dynamics and structuring in pathogen populations, epidemiological multi-strain models have commonly evoked strong immune interactions between strains as the predominant driver. Here, with specific reference to dengue, we show how spatially explicit, multi-strain systems can exhibit all of the described epidemiological dynamics even in the absence of immune competition. Instead, amplification of natural stochastic differences in disease transmission, can give rise to persistent oscillations comprising semi-regular epidemic outbreaks and sequential dominance of dengue's four serotypes. Not only can this mechanism explain observed differences in serotype and disease distributions between neighbouring geographical areas, it also has important implications for inferring the nature and epidemiological consequences of immune mediated competition in multi-strain pathogen systems.A Comprehensive Survey of Small-Molecule Binding Pockets in Proteins [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Mu Gao, Jeffrey Skolnick
Many biological activities originate from interactions between small-molecule ligands and their protein targets. A detailed structural and physico-chemical characterization of these interactions could significantly deepen our understanding of protein function and facilitate drug design. Here, we present a large-scale study on a non-redundant set of about 20,000 known ligand-binding sites, or pockets, of proteins. We find that the structural space of protein pockets is crowded, likely complete, and may be represented by about 1,000 pocket shapes. Correspondingly, the growth rate of novel pockets deposited in the Protein Data Bank has been decreasing steadily over the recent years. Moreover, many protein pockets are promiscuous and interact with ligands of diverse scaffolds. Conversely, many ligands are promiscuous and interact with structurally different pockets. Through a physico-chemical and structural analysis, we provide insights into understanding both pocket promiscuity and ligand promiscuity. Finally, we discuss the implications of our study for the prediction of protein-ligand interactions based on pocket comparison.CAPE: An R Package for Combined Analysis of Pleiotropy and Epistasis [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Anna L. Tyler, Wei Lu, Justin J. Hendrick, Vivek M. Philip, Gregory W. Carter
Contemporary genetic studies are revealing the genetic complexity of many traits in humans and model organisms. Two hallmarks of this complexity are epistasis, meaning gene-gene interaction, and pleiotropy, in which one gene affects multiple phenotypes. Understanding the genetic architecture of complex traits requires addressing these phenomena, but interpreting the biological significance of epistasis and pleiotropy is often difficult. While epistasis reveals dependencies between genetic variants, it is often unclear how the activity of one variant is specifically modifying the other. Epistasis found in one phenotypic context may disappear in another context, rendering the genetic interaction ambiguous. Pleiotropy can suggest either redundant phenotype measures or gene variants that affect multiple biological processes. Here we present an R package, R/cape, which addresses these interpretation ambiguities by implementing a novel method to generate predictive and interpretable genetic networks that influence quantitative phenotypes. R/cape integrates information from multiple related phenotypes to constrain models of epistasis, thereby enhancing the detection of interactions that simultaneously describe all phenotypes. The networks inferred by R/cape are readily interpretable in terms of directed influences that indicate suppressive and enhancing effects of individual genetic variants on other variants, which in turn account for the variance in quantitative traits. We demonstrate the utility of R/cape by analyzing a mouse backcross, thereby discovering novel epistatic interactions influencing phenotypes related to obesity and diabetes. R/cape is an easy-to-use, platform-independent R package and can be applied to data from both genetic screens and a variety of segregating populations including backcrosses, intercrosses, and natural populations. The package is freely available under the GPL-3 license at http://cran.r-project.org/web/packages/cape.Unraveling Adaptation in Eukaryotic Pathways: Lessons from Protocells [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Giovanna De Palo, Robert G. Endres
Eukaryotic adaptation pathways operate within wide-ranging environmental conditions without stimulus saturation. Despite numerous differences in the adaptation mechanisms employed by bacteria and eukaryotes, all require energy consumption. Here, we present two minimal models showing that expenditure of energy by the cell is not essential for adaptation. Both models share important features with large eukaryotic cells: they employ small diffusible molecules and involve receptor subunits resembling highly conserved G-protein cascades. Analyzing the drawbacks of these models helps us understand the benefits of energy consumption, in terms of adjustability of response and adaptation times as well as separation of cell-external sensing and cell-internal signaling. Our work thus sheds new light on the evolution of adaptation mechanisms in complex systems.The Molecular Mechanism of Ion-Dependent Gating in Secondary Transporters [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Chunfeng Zhao, Sergei Yu. Noskov
LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate's binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.Keeping up with the Joneses: Interpersonal Prediction Errors and the Correlation of Behavior in a Tandem Sequential Choice Task [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Terry Lohrenz, Meghana Bhatt, Nathan Apple, P. Read Montague
In many settings, copying, learning from or assigning value to group behavior is rational because such behavior can often act as a proxy for valuable returns. However, such herd behavior can also be pathologically misleading by coaxing individuals into behaviors that are otherwise irrational and it may be one source of the irrational behaviors underlying market bubbles and crashes. Using a two-person tandem investment game, we sought to examine the neural and behavioral responses of herd instincts in situations stripped of the incentive to be influenced by the choices of one's partner. We show that the investments of the two subjects correlate over time if they are made aware of their partner's choices even though these choices have no impact on either player's earnings. We computed an “interpersonal prediction error”, the difference between the investment decisions of the two subjects after each choice. BOLD responses in the striatum, implicated in valuation and action selection, were highly correlated with this interpersonal prediction error. The revelation of the partner's investment occurred after all useful information about the market had already been revealed. This effect was confirmed in two separate experiments where the impact of the time of revelation of the partner's choice was tested at 2 seconds and 6 seconds after a subject's choice; however, the effect was absent in a control condition with a computer partner. These findings strongly support the existence of mechanisms that drive correlated behavior even in contexts where there is no explicit advantage to do so.Molecular Mechanical Differences between Isoforms of Contractile Actin in the Presence of Isoforms of Smooth Muscle Tropomyosin [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Lennart Hilbert, Genevieve Bates, Horia N. Roman, Jenna L. Blumenthal, Nedjma B. Zitouni, Apolinary Sobieszek, Michael C. Mackey, Anne-Marie Lauzon
The proteins involved in smooth muscle's molecular contractile mechanism – the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin – are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for -actin (A), -actin-tropomyosin- (A-Tm), -actin-tropomyosin- (A-Tm), -actin (A), -actin-tropomyosin- (A-Tm), and -actin-tropomoysin- (A-Tm). Actin sliding analysis with our specifically developed video analysis software followed by statistical assessment (Bootstrapped Principal Component Analysis) indicated that the in vitro motility of A, A, and A-Tm is not distinguishable. Compared to these three ‘baseline conditions’, statistically significant differences () were: A-Tm – actin sliding velocity increased 1.12-fold, A-Tm – motile fraction decreased to 0.96-fold, stop time elevated 1.6-fold, A-Tm – run time elevated 1.7-fold. We constructed a mathematical model, simulated actin sliding data, and adjusted the kinetic parameters so as to mimic the experimentally observed differences: A-Tm – myosin binding to actin, the main, and the secondary myosin power stroke are accelerated, A-Tm – mechanical coupling between myosins is stronger, A-Tm – the secondary power stroke is decelerated and mechanical coupling between myosins is weaker. In summary, our results explain the different regulatory effects that specific combinations of actin and smooth muscle tropomyosin have on smooth muscle actin-myosin interaction kinetics.Emergence of Metastable State Dynamics in Interconnected Cortical Networks with Propagation Delays [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Katrina M. Kutchko, Flavio Fröhlich
The importance of the large number of thin-diameter and unmyelinated axons that connect different cortical areas is unknown. The pronounced propagation delays in these axons may prevent synchronization of cortical networks and therefore hinder efficient information integration and processing. Yet, such global information integration across cortical areas is vital for higher cognitive function. We hypothesized that delays in communication between cortical areas can disrupt synchronization and therefore enhance the set of activity trajectories and computations interconnected networks can perform. To evaluate this hypothesis, we studied the effect of long-range cortical projections with propagation delays in interconnected large-scale cortical networks that exhibited spontaneous rhythmic activity. Long-range connections with delays caused the emergence of metastable, spatio-temporally distinct activity states between which the networks spontaneously transitioned. Interestingly, the observed activity patterns correspond to macroscopic network dynamics such as globally synchronized activity, propagating wave fronts, and spiral waves that have been previously observed in neurophysiological recordings from humans and animal models. Transient perturbations with simulated transcranial alternating current stimulation (tACS) confirmed the multistability of the interconnected networks by switching the networks between these metastable states. Our model thus proposes that slower long-range connections enrich the landscape of activity states and represent a parsimonious mechanism for the emergence of multistability in cortical networks. These results further provide a mechanistic link between the known deficits in connectivity and cortical state dynamics in neuropsychiatric illnesses such as schizophrenia and autism, as well as suggest non-invasive brain stimulation as an effective treatment for these illnesses.Attention-Dependent Modulation of Cortical Taste Circuits Revealed by Granger Causality with Signal-Dependent Noise [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Qiang Luo, Tian Ge, Fabian Grabenhorst, Jianfeng Feng, Edmund T. Rolls
We show, for the first time, that in cortical areas, for example the insular, orbitofrontal, and lateral prefrontal cortex, there is signal-dependent noise in the fMRI blood-oxygen level dependent (BOLD) time series, with the variance of the noise increasing approximately linearly with the square of the signal. Classical Granger causal models are based on autoregressive models with time invariant covariance structure, and thus do not take this signal-dependent noise into account. To address this limitation, here we describe a Granger causal model with signal-dependent noise, and a novel, likelihood ratio test for causal inferences. We apply this approach to the data from an fMRI study to investigate the source of the top-down attentional control of taste intensity and taste pleasantness processing. The Granger causality with signal-dependent noise analysis reveals effects not identified by classical Granger causal analysis. In particular, there is a top-down effect from the posterior lateral prefrontal cortex to the insular taste cortex during attention to intensity but not to pleasantness, and there is a top-down effect from the anterior and posterior lateral prefrontal cortex to the orbitofrontal cortex during attention to pleasantness but not to intensity. In addition, there is stronger forward effective connectivity from the insular taste cortex to the orbitofrontal cortex during attention to pleasantness than during attention to intensity. These findings indicate the importance of explicitly modeling signal-dependent noise in functional neuroimaging, and reveal some of the processes involved in a biased activation theory of selective attention.Properties of MHC Class I Presented Peptides That Enhance Immunogenicity [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Jorg J. A. Calis, Matt Maybeno, Jason A. Greenbaum, Daniela Weiskopf, Aruna D. De Silva, Alessandro Sette, Can Keşmir, Bjoern Peters
T-cells have to recognize peptides presented on MHC molecules to be activated and elicit their effector functions. Several studies demonstrate that some peptides are more immunogenic than others and therefore more likely to be T-cell epitopes. We set out to determine which properties cause such differences in immunogenicity. To this end, we collected and analyzed a large set of data describing the immunogenicity of peptides presented on various MHC-I molecules. Two main conclusions could be drawn from this analysis: First, in line with previous observations, we showed that positions P4–6 of a presented peptide are more important for immunogenicity. Second, some amino acids, especially those with large and aromatic side chains, are associated with immunogenicity. This information was combined into a simple model that was used to demonstrate that immunogenicity is, to a certain extent, predictable. This model (made available at http://tools.iedb.org/immunogenicity/) was validated with data from two independent epitope discovery studies. Interestingly, with this model we could show that T-cells are equipped to better recognize viral than human (self) peptides. After the past successful elucidation of different steps in the MHC-I presentation pathway, the identification of variables that influence immunogenicity will be an important next step in the investigation of T-cell epitopes and our understanding of cellular immune responses.The Convallis Rule for Unsupervised Learning in Cortical Networks [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Pierre Yger, Kenneth D. Harris
The phenomenology and cellular mechanisms of cortical synaptic plasticity are becoming known in increasing detail, but the computational principles by which cortical plasticity enables the development of sensory representations are unclear. Here we describe a framework for cortical synaptic plasticity termed the “Convallis rule”, mathematically derived from a principle of unsupervised learning via constrained optimization. Implementation of the rule caused a recurrent cortex-like network of simulated spiking neurons to develop rate representations of real-world speech stimuli, enabling classification by a downstream linear decoder. Applied to spike patterns used in in vitro plasticity experiments, the rule reproduced multiple results including and beyond STDP. However STDP alone produced poorer learning performance. The mathematical form of the rule is consistent with a dual coincidence detector mechanism that has been suggested by experiments in several synaptic classes of juvenile neocortex. Based on this confluence of normative, phenomenological, and mechanistic evidence, we suggest that the rule may approximate a fundamental computational principle of the neocortex.The Influence of Synaptic Weight Distribution on Neuronal Population Dynamics [PLOS Computational Biology: New Articles] 2013-10-24 17:00
by Ramakrishnan Iyer, Vilas Menon, Michael Buice, Christof Koch, Stefan Mihalas
The manner in which different distributions of synaptic weights onto cortical neurons shape their spiking activity remains open. To characterize a homogeneous neuronal population, we use the master equation for generalized leaky integrate-and-fire neurons with shot-noise synapses. We develop fast semi-analytic numerical methods to solve this equation for either current or conductance synapses, with and without synaptic depression. We show that its solutions match simulations of equivalent neuronal networks better than those of the Fokker-Planck equation and we compute bounds on the network response to non-instantaneous synapses. We apply these methods to study different synaptic weight distributions in feed-forward networks. We characterize the synaptic amplitude distributions using a set of measures, called tail weight numbers, designed to quantify the preponderance of very strong synapses. Even if synaptic amplitude distributions are equated for both the total current and average synaptic weight, distributions with sparse but strong synapses produce higher responses for small inputs, leading to a larger operating range. Furthermore, despite their small number, such synapses enable the network to respond faster and with more stability in the face of external fluctuations.On the Role of Aggregation Prone Regions in Protein Evolution, Stability, and Enzymatic Catalysis: Insights from Diverse Analyses [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Patrick M. Buck, Sandeep Kumar, Satish K. Singh
The various roles that aggregation prone regions (APRs) are capable of playing in proteins are investigated here via comprehensive analyses of multiple non-redundant datasets containing randomly generated amino acid sequences, monomeric proteins, intrinsically disordered proteins (IDPs) and catalytic residues. Results from this study indicate that the aggregation propensities of monomeric protein sequences have been minimized compared to random sequences with uniform and natural amino acid compositions, as observed by a lower average aggregation propensity and fewer APRs that are shorter in length and more often punctuated by gate-keeper residues. However, evidence for evolutionary selective pressure to disrupt these sequence regions among homologous proteins is inconsistent. APRs are less conserved than average sequence identity among closely related homologues (≥80% sequence identity with a parent) but APRs are more conserved than average sequence identity among homologues that have at least 50% sequence identity with a parent. Structural analyses of APRs indicate that APRs are three times more likely to contain ordered versus disordered residues and that APRs frequently contribute more towards stabilizing proteins than equal length segments from the same protein. Catalytic residues and APRs were also found to be in structural contact significantly more often than expected by random chance. Our findings suggest that proteins have evolved by optimizing their risk of aggregation for cellular environments by both minimizing aggregation prone regions and by conserving those that are important for folding and function. In many cases, these sequence optimizations are insufficient to develop recombinant proteins into commercial products. Rational design strategies aimed at improving protein solubility for biotechnological purposes should carefully evaluate the contributions made by candidate APRs, targeted for disruption, towards protein structure and activity.Reassessing Google Flu Trends Data for Detection of Seasonal and Pandemic Influenza: A Comparative Epidemiological Study at Three Geographic Scales [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Donald R. Olson, Kevin J. Konty, Marc Paladini, Cecile Viboud, Lone Simonsen
The goal of influenza-like illness (ILI) surveillance is to determine the timing, location and magnitude of outbreaks by monitoring the frequency and progression of clinical case incidence. Advances in computational and information technology have allowed for automated collection of higher volumes of electronic data and more timely analyses than previously possible. Novel surveillance systems, including those based on internet search query data like Google Flu Trends (GFT), are being used as surrogates for clinically-based reporting of influenza-like-illness (ILI). We investigated the reliability of GFT during the last decade (2003 to 2013), and compared weekly public health surveillance with search query data to characterize the timing and intensity of seasonal and pandemic influenza at the national (United States), regional (Mid-Atlantic) and local (New York City) levels. We identified substantial flaws in the original and updated GFT models at all three geographic scales, including completely missing the first wave of the 2009 influenza A/H1N1 pandemic, and greatly overestimating the intensity of the A/H3N2 epidemic during the 2012/2013 season. These results were obtained for both the original (2008) and the updated (2009) GFT algorithms. The performance of both models was problematic, perhaps because of changes in internet search behavior and differences in the seasonality, geographical heterogeneity and age-distribution of the epidemics between the periods of GFT model-fitting and prospective use. We conclude that GFT data may not provide reliable surveillance for seasonal or pandemic influenza and should be interpreted with caution until the algorithm can be improved and evaluated. Current internet search query data are no substitute for timely local clinical and laboratory surveillance, or national surveillance based on local data collection. New generation surveillance systems such as GFT should incorporate the use of near-real time electronic health data and computational methods for continued model-fitting and ongoing evaluation and improvement.Integrated Module and Gene-Specific Regulatory Inference Implicates Upstream Signaling Networks [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Sushmita Roy, Stephen Lagree, Zhonggang Hou, James A. Thomson, Ron Stewart, Audrey P. Gasch
Regulatory networks that control gene expression are important in diverse biological contexts including stress response and development. Each gene's regulatory program is determined by module-level regulation (e.g. co-regulation via the same signaling system), as well as gene-specific determinants that can fine-tune expression. We present a novel approach, Modular regulatory network learning with per gene information (MERLIN), that infers regulatory programs for individual genes while probabilistically constraining these programs to reveal module-level organization of regulatory networks. Using edge-, regulator- and module-based comparisons of simulated networks of known ground truth, we find MERLIN reconstructs regulatory programs of individual genes as well or better than existing approaches of network reconstruction, while additionally identifying modular organization of the regulatory networks. We use MERLIN to dissect global transcriptional behavior in two biological contexts: yeast stress response and human embryonic stem cell differentiation. Regulatory modules inferred by MERLIN capture co-regulatory relationships between signaling proteins and downstream transcription factors thereby revealing the upstream signaling systems controlling transcriptional responses. The inferred networks are enriched for regulators with genetic or physical interactions, supporting the inference, and identify modules of functionally related genes bound by the same transcriptional regulators. Our method combines the strengths of per-gene and per-module methods to reveal new insights into transcriptional regulation in stress and development.Model of Fission Yeast Cell Shape Driven by Membrane-Bound Growth Factors and the Cytoskeleton [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Tyler Drake, Dimitrios Vavylonis
Fission yeast serves as a model for how cellular polarization machinery consisting of signaling molecules and the actin and microtubule cytoskeleton regulates cell shape. In this work, we develop mathematical models to investigate how these cells maintain a tubular shape of approximately constant diameter. Many studies identify active Cdc42, found in a cap at the inner membrane of growing cell tips, as an important regulator of local cell wall remodeling, likely through control of exocyst tethering and the targeting of other polarity-enhancing structures. First, we show that a computational model with Cdc42-dependent local cell wall remodeling under turgor pressure predicts a relationship between spatial extent of growth signal and cell diameter that is in agreement with prior experiments. Second, we model the consequences of feedback between cell shape and distribution of Cdc42 growth signal at cell tips. We show that stability of cell diameter over successive cell divisions places restrictions on their mutual dependence. We argue that simple models where the spatial extent of the tip growth signal relies solely on geometrical alignment of confined microtubules might lead to unstable width regulation. Third, we study a computational model that combines a growth signal distributed over a characteristic length scale (as, for example, by a reaction-diffusion mechanism) with an axis-sensing microtubules system that places landmarks at positions where microtubule tips touch the cortex. A two-dimensional implementation of this model leads to stable cell diameter for a wide range of parameters. Changes to the parameters of this model reproduce straight, bent, and bulged cell shapes, and we discuss how this model is consistent with other observed cell shapes in mutants. Our work provides an initial quantitative framework for understanding the regulation of cell shape in fission yeast, and a scaffold for understanding this process on a more molecular level in the future.Dynamic Change of Global and Local Information Processing in Propofol-Induced Loss and Recovery of Consciousness [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Martin M. Monti, Evan S. Lutkenhoff, Mikail Rubinov, Pierre Boveroux, Audrey Vanhaudenhuyse, Olivia Gosseries, Marie-Aurélie Bruno, Quentin Noirhomme, Mélanie Boly, Steven Laureys
Whether unique to humans or not, consciousness is a central aspect of our experience of the world. The neural fingerprint of this experience, however, remains one of the least understood aspects of the human brain. In this paper we employ graph-theoretic measures and support vector machine classification to assess, in 12 healthy volunteers, the dynamic reconfiguration of functional connectivity during wakefulness, propofol-induced sedation and loss of consciousness, and the recovery of wakefulness. Our main findings, based on resting-state fMRI, are three-fold. First, we find that propofol-induced anesthesia does not bear differently on long-range versus short-range connections. Second, our multi-stage design dissociated an initial phase of thalamo-cortical and cortico-cortical hyperconnectivity, present during sedation, from a phase of cortico-cortical hypoconnectivity, apparent during loss of consciousness. Finally, we show that while clustering is increased during loss of consciousness, as recently suggested, it also remains significantly elevated during wakefulness recovery. Conversely, the characteristic path length of brain networks (i.e., the average functional distance between any two regions of the brain) appears significantly increased only during loss of consciousness, marking a decrease of global information-processing efficiency uniquely associated with unconsciousness. These findings suggest that propofol-induced loss of consciousness is mainly tied to cortico-cortical and not thalamo-cortical mechanisms, and that decreased efficiency of information flow is the main feature differentiating the conscious from the unconscious brain.Analysis of Initial Cell Spreading Using Mechanistic Contact Formulations for a Deformable Cell Model [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Tim Odenthal, Bart Smeets, Paul Van Liedekerke, Engelbert Tijskens, Hans Van Oosterwyck, Herman Ramon
Adhesion governs to a large extent the mechanical interaction between a cell and its microenvironment. As initial cell spreading is purely adhesion driven, understanding this phenomenon leads to profound insight in both cell adhesion and cell-substrate interaction. It has been found that across a wide variety of cell types, initial spreading behavior universally follows the same power laws. The simplest cell type providing this scaling of the radius of the spreading area with time are modified red blood cells (RBCs), whose elastic responses are well characterized. Using a mechanistic description of the contact interaction between a cell and its substrate in combination with a deformable RBC model, we are now able to investigate in detail the mechanisms behind this universal power law. The presented model suggests that the initial slope of the spreading curve with time results from a purely geometrical effect facilitated mainly by dissipation upon contact. Later on, the spreading rate decreases due to increasing tension and dissipation in the cell's cortex as the cell spreads more and more. To reproduce this observed initial spreading, no irreversible deformations are required. Since the model created in this effort is extensible to more complex cell types and can cope with arbitrarily shaped, smooth mechanical microenvironments of the cells, it can be useful for a wide range of investigations where forces at the cell boundary play a decisive role.Reconstructing the Genomic Content of Microbiome Taxa through Shotgun Metagenomic Deconvolution [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Rogan Carr, Shai S. Shen-Orr, Elhanan Borenstein
Metagenomics has transformed our understanding of the microbial world, allowing researchers to bypass the need to isolate and culture individual taxa and to directly characterize both the taxonomic and gene compositions of environmental samples. However, associating the genes found in a metagenomic sample with the specific taxa of origin remains a critical challenge. Existing binning methods, based on nucleotide composition or alignment to reference genomes allow only a coarse-grained classification and rely heavily on the availability of sequenced genomes from closely related taxa. Here, we introduce a novel computational framework, integrating variation in gene abundances across multiple samples with taxonomic abundance data to deconvolve metagenomic samples into taxa-specific gene profiles and to reconstruct the genomic content of community members. This assembly-free method is not bounded by various factors limiting previously described methods of metagenomic binning or metagenomic assembly and represents a fundamentally different approach to metagenomic-based genome reconstruction. An implementation of this framework is available at http://elbo.gs.washington.edu/software.html. We first describe the mathematical foundations of our framework and discuss considerations for implementing its various components. We demonstrate the ability of this framework to accurately deconvolve a set of metagenomic samples and to recover the gene content of individual taxa using synthetic metagenomic samples. We specifically characterize determinants of prediction accuracy and examine the impact of annotation errors on the reconstructed genomes. We finally apply metagenomic deconvolution to samples from the Human Microbiome Project, successfully reconstructing genus-level genomic content of various microbial genera, based solely on variation in gene count. These reconstructed genera are shown to correctly capture genus-specific properties. With the accumulation of metagenomic data, this deconvolution framework provides an essential tool for characterizing microbial taxa never before seen, laying the foundation for addressing fundamental questions concerning the taxa comprising diverse microbial communities.Dynamic Rendering of the Heterogeneous Cell Response to Anticancer Treatments [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Francesca Falcetta, Monica Lupi, Valentina Colombo, Paolo Ubezio
The antiproliferative response to anticancer treatment is the result of concurrent responses in all cell cycle phases, extending over several cell generations, whose complexity is not captured by current methods. In the proposed experimental/computational approach, the contemporary use of time-lapse live cell microscopy and flow cytometric data supported the computer rendering of the proliferative process through the cell cycle and subsequent generations during/after treatment. The effects of treatments were modelled with modules describing the functional activity of the main pathways causing arrest, repair and cell death in each phase. A framework modelling environment was created, enabling us to apply different types of modules in each phase and test models at the complexity level justified by the available data. We challenged the method with time-course measures taken in parallel with flow cytometry and time-lapse live cell microscopy in X-ray-treated human ovarian cancer cells, spanning a wide range of doses. The most suitable model of the treatment, including the dose-response of each effect, was progressively built, combining modules with a rational strategy and fitting simultaneously all data of different doses and platforms. The final model gave for the first time the complete rendering in silico of the cycling process following X-ray exposure, providing separate and quantitative measures of the dose-dependence of G1, S and G2M checkpoint activities in subsequent generations, reconciling known effects of ionizing radiations and new insights in a unique scenario.Prediction of the P. falciparum Target Space Relevant to Malaria Drug Discovery [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Andreas Spitzmüller, Jordi Mestres
Malaria is still one of the most devastating infectious diseases, affecting hundreds of millions of patients worldwide. Even though there are several established drugs in clinical use for malaria treatment, there is an urgent need for new drugs acting through novel mechanisms of action due to the rapid development of resistance. Resistance emerges when the parasite manages to mutate the sequence of the drug targets to the extent that the protein can still perform its function in the parasite but can no longer be inhibited by the drug, which then becomes almost ineffective. The design of a new generation of malaria drugs targeting multiple essential proteins would make it more difficult for the parasite to develop full resistance without lethally disrupting some of its vital functions. The challenge is then to identify which set of Plasmodium falciparum proteins, among the millions of possible combinations, can be targeted at the same time by a given chemotype. To do that, we predicted first the targets of the close to 20,000 antimalarial hits identified recently in three independent phenotypic screening campaigns. All targets predicted were then projected onto the genome of P. falciparum using orthologous relationships. A total of 226 P. falciparum proteins were predicted to be hit by at least one compound, of which 39 were found to be significantly enriched by the presence and degree of affinity of phenotypically active compounds. The analysis of the chemically compatible target combinations containing at least one of those 39 targets led to the identification of a priority set of 64 multi-target profiles that can set the ground for a new generation of more robust malaria drugs.dPeak: High Resolution Identification of Transcription Factor Binding Sites from PET and SET ChIP-Seq Data [PLOS Computational Biology: New Articles] 2013-10-17 17:00
by Dongjun Chung, Dan Park, Kevin Myers, Jeffrey Grass, Patricia Kiley, Robert Landick, Sündüz Keleş
Chromatin immunoprecipitation followed by high throughput sequencing (ChIP-Seq) has been successfully used for genome-wide profiling of transcription factor binding sites, histone modifications, and nucleosome occupancy in many model organisms and humans. Because the compact genomes of prokaryotes harbor many binding sites separated by only few base pairs, applications of ChIP-Seq in this domain have not reached their full potential. Applications in prokaryotic genomes are further hampered by the fact that well studied data analysis methods for ChIP-Seq do not result in a resolution required for deciphering the locations of nearby binding events. We generated single-end tag (SET) and paired-end tag (PET) ChIP-Seq data for factor in Escherichia coli (E. coli). Direct comparison of these datasets revealed that although PET assay enables higher resolution identification of binding events, standard ChIP-Seq analysis methods are not equipped to utilize PET-specific features of the data. To address this problem, we developed dPeak as a high resolution binding site identification (deconvolution) algorithm. dPeak implements a probabilistic model that accurately describes ChIP-Seq data generation process for both the SET and PET assays. For SET data, dPeak outperforms or performs comparably to the state-of-the-art high-resolution ChIP-Seq peak deconvolution algorithms such as PICS, GPS, and GEM. When coupled with PET data, dPeak significantly outperforms SET-based analysis with any of the current state-of-the-art methods. Experimental validations of a subset of dPeak predictions from PET ChIP-Seq data indicate that dPeak can estimate locations of binding events with as high as to resolution. Applications of dPeak to ChIP-Seq data in E. coli under aerobic and anaerobic conditions reveal closely located promoters that are differentially occupied and further illustrate the importance of high resolution analysis of ChIP-Seq data.A Simple Rule for Dendritic Spine and Axonal Bouton Formation Can Account for Cortical Reorganization after Focal Retinal Lesions [PLOS Computational Biology: New Articles] 2013-10-10 17:00
by Markus Butz, Arjen van Ooyen
Lasting alterations in sensory input trigger massive structural and functional adaptations in cortical networks. The principles governing these experience-dependent changes are, however, poorly understood. Here, we examine whether a simple rule based on the neurons' need for homeostasis in electrical activity may serve as driving force for cortical reorganization. According to this rule, a neuron creates new spines and boutons when its level of electrical activity is below a homeostatic set-point and decreases the number of spines and boutons when its activity exceeds this set-point. In addition, neurons need a minimum level of activity to form spines and boutons. Spine and bouton formation depends solely on the neuron's own activity level, and synapses are formed by merging spines and boutons independently of activity. Using a novel computational model, we show that this simple growth rule produces neuron and network changes as observed in the visual cortex after focal retinal lesions. In the model, as in the cortex, the turnover of dendritic spines was increased strongest in the center of the lesion projection zone, while axonal boutons displayed a marked overshoot followed by pruning. Moreover, the decrease in external input was compensated for by the formation of new horizontal connections, which caused a retinotopic remapping. Homeostatic regulation may provide a unifying framework for understanding cortical reorganization, including network repair in degenerative diseases or following focal stroke.Feature Selection Methods for Identifying Genetic Determinants of Host Species in RNA Viruses [PLOS Computational Biology: New Articles] 2013-10-10 17:00
by Ricardo Aguas, Neil M. Ferguson
Despite environmental, social and ecological dependencies, emergence of zoonotic viruses in human populations is clearly also affected by genetic factors which determine cross-species transmission potential. RNA viruses pose an interesting case study given their mutation rates are orders of magnitude higher than any other pathogen – as reflected by the recent emergence of SARS and Influenza for example. Here, we show how feature selection techniques can be used to reliably classify viral sequences by host species, and to identify the crucial minority of host-specific sites in pathogen genomic data. The variability in alleles at those sites can be translated into prediction probabilities that a particular pathogen isolate is adapted to a given host. We illustrate the power of these methods by: 1) identifying the sites explaining SARS coronavirus differences between human, bat and palm civet samples; 2) showing how cross species jumps of rabies virus among bat populations can be readily identified; and 3) de novo identification of likely functional influenza host discriminant markers.Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Despite numerous studies on specific sumoylated transcriptional regulators, the global role of SUMO on chromatin in relation to transcription regulation remains largely unknown. Here, we determined the genome-wide localization of SUMO1 and SUMO2/3, as well as of UBC9 (encoded by UBE2I) and PIASY (encoded by PIAS4), two markers for active sumoylation, along with Pol II and histone marks in proliferating versus senescent human fibroblasts together with gene expression profiling. We found that, whereas SUMO alone is widely distributed over the genome with strong association at active promoters, active sumoylation occurs most prominently at promoters of histone and protein biogenesis genes, as well as Pol I rRNAs and Pol III tRNAs. Remarkably, these four classes of genes are up-regulated by inhibition of sumoylation, indicating that SUMO normally acts to restrain their expression. In line with this finding, sumoylation-deficient cells show an increase in both cell size and global protein levels. Strikingly, we found that in senescent cells, the SUMO machinery is selectively retained at histone and tRNA gene clusters, whereas it is massively released from all other unique chromatin regions. These data, which reveal the highly dynamic nature of the SUMO landscape, suggest that maintenance of a repressive environment at histone and tRNA loci is a hallmark of the senescent state. The approach taken in our study thus permitted the identification of a common biological output and uncovered hitherto unknown functions for active sumoylation at chromatin as a key mechanism that, in dynamically marking chromatin by a simple modifier, orchestrates concerted transcriptional regulation of a network of genes essential for cell growth and proliferation.
Lamin A/C-promoter interactions specify chromatin state-dependent transcription outcomes [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
The nuclear lamina is implicated in the organization of the eukaryotic nucleus. Association of nuclear lamins with the genome occurs through large chromatin domains including mostly, but not exclusively, repressed genes. How lamin interactions with regulatory elements modulate gene expression in different cellular contexts is unknown. We show here that in human adipose tissue stem cells, lamin A/C interacts with distinct spatially restricted subpromoter regions, both within and outside peripheral and intra-nuclear lamin-rich domains. These localized interactions are associated with distinct transcriptional outcomes in a manner dependent on local chromatin modifications. Down-regulation of lamin A/C leads to dissociation of lamin A/C from promoters and remodels repressive and permissive histone modifications by enhancing transcriptional permissiveness, but is not sufficient to elicit gene activation. Adipogenic differentiation resets a large number of lamin-genome associations globally and at subpromoter levels and redefines associated transcription outputs. We propose that lamin A/C acts as a modulator of local gene expression outcome through interaction with adjustable sites on promoters, and that these position-dependent transcriptional readouts may be reset upon differentiation.
GC skew at the 5' and 3' ends of human genes links R-loop formation to epigenetic regulation and transcription termination [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Strand asymmetry in the distribution of guanines and cytosines, measured by GC skew, predisposes DNA sequences toward R-loop formation upon transcription. Previous work revealed that GC skew and R-loop formation associate with a core set of unmethylated CpG island (CGI) promoters in the human genome. Here, we show that GC skew can distinguish four classes of promoters, including three types of CGI promoters, each associated with unique epigenetic and gene ontology signatures. In particular, we identify a strong and a weak class of CGI promoters and show that these loci are enriched in distinct chromosomal territories reflecting the intrinsic strength of their protection against DNA methylation. Interestingly, we show that strong CGI promoters are depleted from the X chromosome while weak CGIs are enriched, a property consistent with the acquisition of DNA methylation during dosage compensation. Furthermore, we identify a third class of CGI promoters based on its unique GC skew profile and show that this gene set is enriched for Polycomb group targets. Lastly, we show that nearly 2000 genes harbor GC skew at their 3' ends and that these genes are preferentially located in gene-dense regions and tend to be closely arranged. Genomic profiling of R-loops accordingly showed that a large proportion of genes with terminal GC skew form R-loops at their 3' ends, consistent with a role for these structures in permitting efficient transcription termination. Altogether, we show that GC skew and R-loop formation offer significant insights into the epigenetic regulation, genomic organization, and function of human genes.
De novo DNA demethylation and noncoding transcription define active intergenic regulatory elements [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Deep sequencing of mammalian DNA methylomes has uncovered a previously unpredicted number of discrete hypomethylated regions in intergenic space (iHMRs). Here, we combined whole-genome bisulfite sequencing data with extensive gene expression and chromatin-state data to define functional classes of iHMRs, and to reconstruct the dynamics of their establishment in a developmental setting. Comparing HMR profiles in embryonic stem and primary blood cells, we show that iHMRs mark an exclusive subset of active DNase hypersensitive sites (DHS), and that both developmentally constitutive and cell-type-specific iHMRs display chromatin states typical of distinct regulatory elements. We also observe that iHMR changes are more predictive of nearby gene activity than the promoter HMR itself, and that expression of noncoding RNAs within the iHMR accompanies full activation and complete demethylation of mature B cell enhancers. Conserved sequence features corresponding to iHMR transcript start sites, including a discernible TATA motif, suggest a conserved, functional role for transcription in these regions. Similarly, we explored both primate-specific and human population variation at iHMRs, finding that while enhancer iHMRs are more variable in sequence and methylation status than any other functional class, conservation of the TATA box is highly predictive of iHMR maintenance, reflecting the impact of sequence plasticity and transcriptional signals on iHMR establishment. Overall, our analysis allowed us to construct a three-step timeline in which (1) intergenic DHS are pre-established in the stem cell, (2) partial demethylation of blood-specific intergenic DHSs occurs in blood progenitors, and (3) complete iHMR formation and transcription coincide with enhancer activation in lymphoid-specified cells.
Frac-seq reveals isoform-specific recruitment to polyribosomes [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Pre-mRNA splicing is required for the accurate expression of virtually all human protein coding genes. However, splicing also plays important roles in coordinating subsequent steps of pre-mRNA processing such as polyadenylation and mRNA export. Here, we test the hypothesis that nuclear pre-mRNA processing influences the polyribosome association of alternative mRNA isoforms. By comparing isoform ratios in cytoplasmic and polyribosomal extracts, we determined that the alternative products of ~30% (597/1954) of mRNA processing events are differentially partitioned between these subcellular fractions. Many of the events exhibiting isoform-specific polyribosome association are highly conserved across mammalian genomes, underscoring their possible biological importance. We find that differences in polyribosome association may be explained, at least in part by the observation that alternative splicing alters the cis-regulatory landscape of mRNAs isoforms. For example, inclusion or exclusion of upstream open reading frames (uORFs) in the 5'UTR as well as Alu-elements and microRNA target sites in the 3'UTR have a strong influence on polyribosome association of alternative mRNA isoforms. Taken together, our data demonstrate for the first time the potential link between alternative splicing and translational control of the resultant mRNA isoforms.
Genome-wide and parental allele-specific analysis of CTCF and cohesin DNA binding in mouse brain reveals a tissue-specific binding pattern and an association with imprinted differentially methylated regions [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
DNA binding factors are essential for regulating gene expression. CTCF and cohesin are DNA binding factors with central roles in chromatin organization and gene expression. We determined the sites of CTCF and cohesin binding to DNA in mouse brain, genome wide and in an allele-specific manner with high read-depth ChIP-seq. By comparing our results with existing data for mouse liver and embryonic stem (ES) cells, we investigated the tissue specificity of CTCF binding sites. ES cells have fewer unique CTCF binding sites occupied than liver and brain, consistent with a ground-state pattern of CTCF binding that is elaborated during differentiation. CTCF binding sites without the canonical consensus motif were highly tissue specific. In brain, a third of CTCF and cohesin binding sites coincide, consistent with the potential for many interactions between cohesin and CTCF but also many instances of independent action. In the context of genomic imprinting, CTCF and/or cohesin bind to a majority but not all differentially methylated regions, with preferential binding to the unmethylated parental allele. Whether the parental allele-specific methylation was established in the parental germlines or post-fertilization in the embryo is not a determinant in CTCF or cohesin binding. These findings link CTCF and cohesin with the control regions of a subset of imprinted genes, supporting the notion that imprinting control is mechanistically diverse.
Global analyses of UPF1 binding and function reveal expanded scope of nonsense-mediated mRNA decay [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
UPF1 is a DNA/RNA helicase with essential roles in nonsense-mediated mRNA decay (NMD) and embryonic development. How UPF1 regulates target abundance and the relationship between NMD and embryogenesis are not well understood. To explore how NMD shapes the embryonic transcriptome, we integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). We identified over 200 direct UPF1 binding targets using crosslinking/immunoprecipitation-sequencing (CLIP-seq) and revealed a repression pathway that involves 3' UTR binding by UPF1 and translation but is independent of canonical targeting features involving 3' UTR length and stop codon placement. Interestingly, NMD targeting of this set of mRNAs occurs in other mouse tissues and is conserved in human. We also show, using ribosome footprint profiling, that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression. Together, our results identify novel NMD determinants and targets and provide context for understanding the impact of UPF1 and NMD on the mESC transcriptome.
The maize methylome influences mRNA splice sites and reveals widespread paramutation-like switches guided by small RNA [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
The maize genome, with its large complement of transposons and repeats, is a paradigm for the study of epigenetic mechanisms such as paramutation and imprinting. Here, we present the genome-wide map of cytosine methylation for two maize inbred lines, B73 and Mo17. CG (65%) and CHG (50%) methylation (where H = A, C, or T) is highest in transposons, while CHH (5%) methylation is likely guided by 24-nt, but not 21-nt, small interfering RNAs (siRNAs). Correlations with methylation patterns suggest that CG methylation in exons (8%) may deter insertion of Mutator transposon insertion, while CHG methylation at splice acceptor sites may inhibit RNA splicing. Using the methylation map as a guide, we used low-coverage sequencing to show that parental methylation differences are inherited by recombinant inbred lines. However, frequent methylation switches, guided by siRNA, persist for up to eight generations, suggesting that epigenetic inheritance resembling paramutation is much more common than previously supposed. The methylation map will provide an invaluable resource for epigenetic studies in maize.
Epigenome-wide inheritance of cytosine methylation variants in a recombinant inbred population [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Cytosine DNA methylation is one avenue for passing information through cell divisions. Here, we present epigenomic analyses of soybean recombinant inbred lines (RILs) and their parents. Identification of differentially methylated regions (DMRs) revealed that DMRs mostly cosegregated with the genotype from which they were derived, but examples of the uncoupling of genotype and epigenotype were identified. Linkage mapping of methylation states assessed from whole-genome bisulfite sequencing of 83 RILs uncovered widespread evidence for local methylQTL. This epigenomics approach provides a comprehensive study of the patterns and heritability of methylation variants in a complex genetic population over multiple generations, paving the way for understanding how methylation variants contribute to phenotypic variation.
Multiple RNA recognition patterns during microRNA biogenesis in plants [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
MicroRNAs (miRNAs) derive from longer precursors with fold-back structures. While animal miRNA precursors have homogenous structures, plant precursors comprise a collection of fold-backs with variable size and shape. Here, we design an approach to systematically analyze miRNA processing intermediates and characterize the biogenesis of most of the evolutionarily conserved miRNAs present in Arabidopsis thaliana. We found that plant miRNAs are processed by four mechanisms, depending on the sequential direction of the processing machinery and the number of cuts required to release the miRNA. Classification of the precursors according to their processing mechanism revealed specific structural determinants for each group. We found that the complexity of the miRNA processing pathways occurs in both ancient and evolutionarily young sequences and that members of the same family can be processed in different ways. We observed that different structural determinants compete for the processing machinery and that alternative miRNAs can be generated from a single precursor. The results provide an explanation for the structural diversity of miRNA precursors in plants and new insights toward the understanding of the biogenesis of small RNAs.
DNA damage induces targeted, genome-wide variation of poly(A) sites in budding yeast [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Systemic response to DNA damage and other stresses is a complex process that includes changes in the regulation and activity of nearly all stages of gene expression. One gene regulatory mechanism used by eukaryotes is selection among alternative transcript isoforms that differ in polyadenylation [poly(A)] sites, resulting in changes either to the coding sequence or to portions of the 3' UTR that govern translation, stability, and localization. To determine the extent to which this means of regulation is used in response to DNA damage, we conducted a global analysis of poly(A) site usage in Saccharomyces cerevisiae after exposure to the UV mimetic, 4-nitroquinoline 1-oxide (4NQO). Two thousand thirty-one genes were found to have significant variation in poly(A) site distributions following 4NQO treatment, with a strong bias toward loss of short transcripts, including many with poly(A) sites located within the protein coding sequence (CDS). We further explored one possible mechanism that could contribute to the widespread differences in mRNA isoforms. The change in poly(A) site profile was associated with an inhibition of cleavage and polyadenylation in cell extract and a decrease in the levels of several key subunits in the mRNA 3'-end processing complex. Sequence analysis identified differences in the cis-acting elements that flank putatively suppressed and enhanced poly(A) sites, suggesting a mechanism that could discriminate between variable and constitutive poly(A) sites. Our analysis indicates that variation in mRNA length is an important part of the regulatory response to DNA damage.
Meta-analyses of studies of the human microbiota [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
Our body habitat-associated microbial communities are of intense research interest because of their influence on human health. Because many studies of the microbiota are based on the same bacterial 16S ribosomal RNA (rRNA) gene target, they can, in principle, be compared to determine the relative importance of different disease/physiologic/developmental states. However, differences in experimental protocols used may produce variation that outweighs biological differences. By comparing 16S rRNA gene sequences generated from diverse studies of the human microbiota using the QIIME database, we found that variation in composition of the microbiota across different body sites was consistently larger than technical variability across studies. However, samples from different studies of the Western adult fecal microbiota generally clustered by study, and the 16S rRNA target region, DNA extraction technique, and sequencing platform produced systematic biases in observed diversity that could obscure biologically meaningful compositional differences. In contrast, systematic compositional differences in the fecal microbiota that occurred with age and between Western and more agrarian cultures were great enough to outweigh technical variation. Furthermore, individuals with ileal Crohn's disease and in their third trimester of pregnancy often resembled infants from different studies more than controls from the same study, indicating parallel compositional attributes of these distinct developmental/physiological/disease states. Together, these results show that cross-study comparisons of human microbiota are valuable when the studied parameter has a large effect size, but studies of more subtle effects on the human microbiota require carefully selected control populations and standardized protocols.
Sympatric chimpanzees and gorillas harbor convergent gut microbial communities [RESEARCH] [Genome Research current issue] 2013-10-01 10:01
The gut microbial communities within great apes have been shown to reflect the phylogenetic history of their hosts, indicating codiversification between great apes and their gut microbiota over evolutionary timescales. But because the great apes examined to date represent geographically isolated populations whose diets derive from different sources, it is unclear whether this pattern of codiversification has resulted from a long history of coadaptation between microbes and hosts (heritable factors) or from the ecological and geographic separation among host species (environmental factors). To evaluate the relative influences of heritable and environmental factors on the evolution of the great ape gut microbiota, we assayed the gut communities of sympatric and allopatric populations of chimpanzees, bonobos, and gorillas residing throughout equatorial Africa. Comparisons of these populations revealed that the gut communities of different host species can always be distinguished from one another but that the gut communities of sympatric chimpanzees and gorillas have converged in terms of community composition, sharing on average 53% more bacterial phylotypes than the gut communities of allopatric hosts. Host environment, independent of host genetics and evolutionary history, shaped the distribution of bacterial phylotypes across the Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria, the four most common phyla of gut bacteria. Moreover, the specific patterns of phylotype sharing among hosts suggest that chimpanzees living in sympatry with gorillas have acquired bacteria from gorillas. These results indicate that geographic isolation between host species has promoted the evolutionary differentiation of great ape gut bacterial communities.
Pathoscope: Species identification and strain attribution with unassembled sequencing data [METHOD] [Genome Research current issue] 2013-10-01 10:01
Emerging next-generation sequencing technologies have revolutionized the collection of genomic data for applications in bioforensics, biosurveillance, and for use in clinical settings. However, to make the most of these new data, new methodology needs to be developed that can accommodate large volumes of genetic data in a computationally efficient manner. We present a statistical framework to analyze raw next-generation sequence reads from purified or mixed environmental or targeted infected tissue samples for rapid species identification and strain attribution against a robust database of known biological agents. Our method, Pathoscope, capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality, and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample and considers cases when the sample species/strain is not in the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for multiple alignment steps, extensive homology searches, or genome assembly—which are time-consuming and labor-intensive steps. We demonstrate the utility of our approach on genomic data from purified and in silico "environmental" samples from known bacterial agents impacting human health for accuracy assessment and comparison with other approaches.
Integrated detection of natural antisense transcripts using strand-specific RNA sequencing data [METHOD] [Genome Research current issue] 2013-10-01 10:01
Pairs of RNA molecules transcribed from partially or entirely complementary loci are called cis-natural antisense transcripts (cis-NATs), and they play key roles in the regulation of gene expression in many organisms. A promising experimental tool for profiling sense and antisense transcription is strand-specific RNA sequencing (ssRNA-seq). To identify cis-NATs using ssRNA-seq, we developed a new computational method based on a model comparison framework that incorporates the inherent variable efficiency of generating perfectly strand-specific libraries. Applying the method to new ssRNA-seq data from whole-root and cell-type–specific Arabidopsis libraries confirmed most of the known cis-NAT pairs and identified 918 additional cis-NAT pairs. Newly identified cis-NAT pairs are supported by polyadenylation data, alternative splicing patterns, and RT-PCR validation. We found 209 cis-NAT pairs that have opposite expression levels in neighboring cell types, implying cell-type–specific roles for cis-NATs. By integrating a genome-wide epigenetic profile of Arabidopsis, we identified a unique chromatin signature of cis-NATs, suggesting a connection between cis-NAT transcription and chromatin modification in plants. An analysis of small-RNA sequencing data showed that ~4% of cis-NAT pairs produce putative cis-NAT–induced siRNAs. Taken together, our data and analyses illustrate the potential for multifaceted regulatory roles of plant cis-NATs.
Coelacanth genomes reveal signatures for evolutionary transition from water to land [RESOURCES] [Genome Research current issue] 2013-10-01 10:01
Coelacanths are known as "living fossils," as they show remarkable morphological resemblance to the fossil record and belong to the most primitive lineage of living Sarcopterygii (lobe-finned fishes and tetrapods). Coelacanths may be key to elucidating the tempo and mode of evolution from fish to tetrapods. Here, we report the genome sequences of five coelacanths, including four Latimeria chalumnae individuals (three specimens from Tanzania and one from Comoros) and one L. menadoensis individual from Indonesia. These sequences cover two African breeding populations and two known extant coelacanth species. The genome is ~2.74 Gbp and contains a high proportion (~60%) of repetitive elements. The genetic diversity among the individuals was extremely low, suggesting a small population size and/or a slow rate of evolution. We found a substantial number of genes that encode olfactory and pheromone receptors with features characteristic of tetrapod receptors for the detection of airborne ligands. We also found that limb enhancers of bmp7 and gli3, both of which are essential for limb formation, are conserved between coelacanth and tetrapods, but not ray-finned fishes. We expect that some tetrapod-like genes may have existed early in the evolution of primitive Sarcopterygii and were later co-opted to adapt to terrestrial environments. These coelacanth genomes will provide a cornerstone for studies to elucidate how ancestral aquatic vertebrates evolved into terrestrial animals.
The million mutation project: A new approach to genetics in Caenorhabditis elegans [RESOURCES] [Genome Research current issue] 2013-10-01 10:01
We have created a library of 2007 mutagenized Caenorhabditis elegans strains, each sequenced to a target depth of 15-fold coverage, to provide the research community with mutant alleles for each of the worm's more than 20,000 genes. The library contains over 800,000 unique single nucleotide variants (SNVs) with an average of eight nonsynonymous changes per gene and more than 16,000 insertion/deletion (indel) and copy number changes, providing an unprecedented genetic resource for this multicellular organism. To supplement this collection, we also sequenced 40 wild isolates, identifying more than 630,000 unique SNVs and 220,000 indels. Comparison of the two sets demonstrates that the mutant collection has a much richer array of both nonsense and missense mutations than the wild isolate set. We also find a wide range of rDNA and telomere repeat copy number in both sets. Scanning the mutant collection for molecular phenotypes reveals a nonsense suppressor as well as strains with higher levels of indels that harbor mutations in DNA repair genes and strains with abundant males associated with him mutations. All the strains are available through the Caenorhabditis Genetics Center and all the sequence changes have been deposited in WormBase and are available through an interactive website.
Nucleic Acids Research: VOLUME 41 ISSUE 19 2013 [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Nucleic Acids Research - Editorial Board [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Subscriptions [Nucleic Acids Research - recent issues] 2013-10-18 05:10
BlackOPs: increasing confidence in variant detection through mappability filtering [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Identifying variants using high-throughput sequencing data is currently a challenge because true biological variants can be indistinguishable from technical artifacts. One source of technical artifact results from incorrectly aligning experimentally observed sequences to their true genomic origin (‘mismapping’) and inferring differences in mismapped sequences to be true variants. We developed BlackOPs, an open-source tool that simulates experimental RNA-seq and DNA whole exome sequences derived from the reference genome, aligns these sequences by custom parameters, detects variants and outputs a blacklist of positions and alleles caused by mismapping. Blacklists contain thousands of artifact variants that are indistinguishable from true variants and, for a given sample, are expected to be almost completely false positives. We show that these blacklist positions are specific to the alignment algorithm and read length used, and BlackOPs allows users to generate a blacklist specific to their experimental setup. We queried the dbSNP and COSMIC variant databases and found numerous variants indistinguishable from mapping errors. We demonstrate how filtering against blacklist positions reduces the number of potential false variants using an RNA-seq glioblastoma cell line data set. In summary, accounting for mapping-caused variants tuned to experimental setups reduces false positives and, therefore, improves genome characterization by high-throughput sequencing.
Torsionally constrained DNA for single-molecule assays: an efficient, ligation-free method [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Controlled twisting of individual, double-stranded DNA molecules provides a unique method to investigate the enzymes that alter DNA topology. Such twisting requires a single DNA molecule to be torsionally constrained. This constraint is achieved by anchoring the opposite ends of the DNA to two separate surfaces via multiple bonds. The traditional protocol for making such DNA involves a three-way ligation followed by gel purification, a laborious process that often leads to low yield both in the amount of DNA and the fraction of molecules that is torsionally constrained. We developed a simple ligation-free procedure for making torsionally constrained DNA via polymerase chain reaction (PCR). This PCR protocol used two ‘megaprimers’, 400-base-pair long double-stranded DNA that were labelled with either biotin or digoxigenin. We obtained a relatively high yield of gel-purified DNA (~500 ng/100 µl of PCR reaction). The final construct in this PCR-based method contains only one labelled strand in contrast to the traditional construct in which both strands of the DNA are labelled. Nonetheless, we achieved a high yield (84%) of torsionally constrained DNA when measured using an optical-trap-based DNA-overstretching assay. This protocol significantly simplifies the application and adoption of torsionally constrained assays to a wide range of single-molecule systems.
Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring -H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.
In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.
Receptor-mediated delivery of engineered nucleases for genome modification [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.
The sequencing bias relaxed characteristics of Hi-C derived data and implications for chromatin 3D modeling [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The 3D chromatin structure modeling by chromatin interactions derived from Hi-C experiments is significantly challenged by the intrinsic sequencing biases in these experiments. Conventional modeling methods only focus on the bias among different chromatin regions within the same experiment but neglect the bias arising from different experimental sequencing depth. We now show that the regional interaction bias is tightly coupled with the sequencing depth, and we further identify a chromatin structure parameter as the inherent characteristics of Hi-C derived data for chromatin regions. Then we present an approach for chromatin structure prediction capable of relaxing both kinds of sequencing biases by using this identified parameter. This method is validated by intra and inter cell-line comparisons among various chromatin regions for four human cell-lines (K562, GM12878, IMR90 and H1hESC), which shows that the openness of chromatin region is well correlated with chromatin function. This method has been executed by an automatic pipeline (AutoChrom3D) and thus can be conveniently used.
Heterogeneous staining: a tool for studies of how fluorescent dyes affect the physical properties of DNA [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The commonly used fluorescent dye YOYO-1 (YOYO) has, using bulk techniques, been demonstrated to stain DNA heterogeneously at substoichiometric concentrations. We here, using nanofluidic channels and fluorescence microscopy, investigate the heterogeneous staining on the single DNA molecule level and demonstrate that the dye distribution is continuous. The equilibration of YOYO on DNA is extremely slow but can be accelerated by increasing the ionic strength and/or the temperature. Furthermore, we demonstrate how to use the heterogeneous staining as a tool for detailed and time-efficient studies of how fluorescent dyes affect the physical properties of DNA. We show that the relative increase in extension of DNA with increasing amount of YOYO bound is higher at low ionic strengths and also extrapolate the extension of native DNA. Our study reveals important information on how YOYO affects the physical properties of DNA, but it also has broader applications. First, it reveals how cationic intercalators, such as potential DNA drugs, affect DNA under strong confinement. Second, the strategy of using heterogeneous staining is of general use for single molecule studies of DNA interacting with proteins or ligands.
Development of a universal radioactive DNA methyltransferase inhibition test for high-throughput screening and mechanistic studies [Nucleic Acids Research - recent issues] 2013-10-18 05:10
DNA methylation is an important epigenetic mark in eukaryotes, and aberrant pattern of this modification is involved in numerous diseases such as cancers. Interestingly, DNA methylation is reversible and thus is considered a promising therapeutic target. Therefore, there is a need for identifying new small inhibitors of C5 DNA methyltransferases (DNMTs). Despite the development of numerous in vitro DNMT assays, there is a lack of reliable tests suitable for high-throughput screening, which can also give insights into inhibitor mechanisms of action. We developed a new test based on scintillation proximity assay meeting these requirements. After optimizing our assay on human DNMT1 and calibrating it with two known inhibitors, we carried out S-Adenosyl-l-Methionine and DNA competition studies on three inhibitors and were able to determine each mechanism of action. Finally, we showed that our test was applicable to 3 other methyltransferases sources: human DNMT3A, bacterial M.SssI and cellular extracts as well.
Sequence-specific microscopic visualization of DNA methylation status at satellite repeats in individual cell nuclei and chromosomes [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Methylation-specific fluorescence in situ hybridization (MeFISH) was developed for microscopic visualization of DNA methylation status at specific repeat sequences in individual cells. MeFISH is based on the differential reactivity of 5-methylcytosine and cytosine in target DNA for interstrand complex formation with osmium and bipyridine-containing nucleic acids (ICON). Cell nuclei and chromosomes hybridized with fluorescence-labeled ICON probes for mouse major and minor satellite repeats were treated with osmium for crosslinking. After denaturation, fluorescent signals were retained specifically at satellite repeats in wild-type, but not in DNA methyltransferase triple-knockout (negative control) mouse embryonic stem cells. Moreover, using MeFISH, we successfully detected hypomethylated satellite repeats in cells from patients with immunodeficiency, centromeric instability and facial anomalies syndrome and 5-hydroxymethylated satellite repeats in male germ cells, the latter of which had been considered to be unmethylated based on anti-5-methylcytosine antibody staining. MeFISH will be suitable for a wide range of applications in epigenetics research and medical diagnosis.
An integrative characterization of recurrent molecular aberrations in glioblastoma genomes [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults. Decades of investigations and the recent effort of the Cancer Genome Atlas (TCGA) project have mapped many molecular alterations in GBM cells. Alterations on DNAs may dysregulate gene expressions and drive malignancy of tumors. It is thus important to uncover causal and statistical dependency between ‘effector’ molecular aberrations and ‘target’ gene expressions in GBMs. A rich collection of prior studies attempted to combine copy number variation (CNV) and mRNA expression data. However, systematic methods to integrate multiple types of cancer genomic data—gene mutations, single nucleotide polymorphisms, CNVs, DNA methylations, mRNA and microRNA expressions and clinical information—are relatively scarce. We proposed an algorithm to build ‘association modules’ linking effector molecular aberrations and target gene expressions and applied the module-finding algorithm to the integrated TCGA GBM data sets. The inferred association modules were validated by six tests using external information and datasets of central nervous system tumors: (i) indication of prognostic effects among patients; (ii) coherence of target gene expressions; (iii) retention of effector–target associations in external data sets; (iv) recurrence of effector molecular aberrations in GBM; (v) functional enrichment of target genes; and (vi) co-citations between effectors and targets. Modules associated with well-known molecular aberrations of GBM—such as chromosome 7 amplifications, chromosome 10 deletions, EGFR and NF1 mutations—passed the majority of the validation tests. Furthermore, several modules associated with less well-reported molecular aberrations—such as chromosome 11 CNVs, CD40, PLXNB1 and GSTM1 methylations, and mir-21 expressions—were also validated by external information. In particular, modules constituting trans-acting effects with chromosome 11 CNVs and cis-acting effects with chromosome 10 CNVs manifested strong negative and positive associations with survival times in brain tumors. By aligning the information of association modules with the established GBM subclasses based on transcription or methylation levels, we found each subclass possessed multiple concurrent molecular aberrations. Furthermore, the joint molecular characteristics derived from 16 association modules had prognostic power not explained away by the strong biomarker of CpG island methylator phenotypes. Functional and survival analyses indicated that immune/inflammatory responses and epithelial-mesenchymal transitions were among the most important determining processes of prognosis. Finally, we demonstrated that certain molecular aberrations uniquely recurred in GBM but were relatively rare in non-GBM glioma cells. These results justify the utility of an integrative analysis on cancer genomes and provide testable characterizations of driver aberration events in GBM.
Identification of cis-regulatory modules in promoters of human genes exploiting mutual positioning of transcription factors [Nucleic Acids Research - recent issues] 2013-10-18 05:10
In higher organisms, gene regulation is controlled by the interplay of non-random combinations of multiple transcription factors (TFs). Although numerous attempts have been made to identify these combinations, important details, such as mutual positioning of the factors that have an important role in the TF interplay, are still missing. The goal of the present work is in silico mapping of some of such associating factors based on their mutual positioning, using computational screening. We have selected the process of myogenesis as a study case, and we focused on TF combinations involving master myogenic TF Myogenic differentiation (MyoD) with other factors situated at specific distances from it. The results of our work show that some muscle-specific factors occur together with MyoD within the range of ±100 bp in a large number of promoters. We confirm co-occurrence of the MyoD with muscle-specific factors as described in earlier studies. However, we have also found novel relationships of MyoD with other factors not specific for muscle. Additionally, we have observed that MyoD tends to associate with different factors in proximal and distal promoter areas. The major outcome of our study is establishing the genome-wide connection between biological interactions of TFs and close co-occurrence of their binding sites.
Environmental shaping of codon usage and functional adaptation across microbial communities [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Microbial communities represent the largest portion of the Earth’s biomass. Metagenomics projects use high-throughput sequencing to survey these communities and shed light on genetic capabilities that enable microbes to inhabit every corner of the biosphere. Metagenome studies are generally based on (i) classifying and ranking functions of identified genes; and (ii) estimating the phyletic distribution of constituent microbial species. To understand microbial communities at the systems level, it is necessary to extend these studies beyond the species’ boundaries and capture higher levels of metabolic complexity. We evaluated 11 metagenome samples and demonstrated that microbes inhabiting the same ecological niche share common preferences for synonymous codons, regardless of their phylogeny. By exploring concepts of translational optimization through codon usage adaptation, we demonstrated that community-wide bias in codon usage can be used as a prediction tool for lifestyle-specific genes across the entire microbial community, effectively considering microbial communities as meta-genomes. These findings set up a ‘functional metagenomics’ platform for the identification of genes relevant for adaptations of entire microbial communities to environments. Our results provide valuable arguments in defining the concept of microbial species through the context of their interactions within the community.
Systems biology of Ewing sarcoma: a network model of EWS-FLI1 effect on proliferation and apoptosis [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Ewing sarcoma is the second most frequent pediatric bone tumor. In most of the patients, a chromosomal translocation leads to the expression of the EWS-FLI1 chimeric transcription factor that is the major oncogene in this pathology. Relative genetic simplicity of Ewing sarcoma makes it particularly attractive for studying cancer in a systemic manner. Silencing EWS-FLI1 induces cell cycle alteration and ultimately leads to apoptosis, but the exact molecular mechanisms underlying this phenotype are unclear. In this study, a network linking EWS-FLI1 to cell cycle and apoptosis phenotypes was constructed through an original method of network reconstruction. Transcriptome time-series after EWS-FLI1 silencing were used to identify core modulated genes by an original scoring method based on fitting expression profile dynamics curves. Literature data mining was then used to connect these modulated genes into a network. The validity of a subpart of this network was assessed by siRNA/RT-QPCR experiments on four additional Ewing cell lines and confirmed most of the links. Based on the network and the transcriptome data, CUL1 was identified as a new potential target of EWS-FLI1. Altogether, using an original methodology of data integration, we provide the first version of EWS-FLI1 network model of cell cycle and apoptosis regulation.
Upstream mononucleotide A-repeats play a cis-regulatory role in mammals through the DICER1 and Ago proteins [Nucleic Acids Research - recent issues] 2013-10-18 05:10
A-repeats are the simplest form of tandem repeats and are found ubiquitously throughout genomes. These mononucleotide repeats have been widely believed to be non-functional ‘junk’ DNA. However, studies in yeasts suggest that A-repeats play crucial biological functions, and their role in humans remains largely unknown. Here, we showed a non-random pattern of distribution of sense A- and T-repeats within 20 kb around transcription start sites (TSSs) in the human genome. Different distributions of these repeats are observed upstream and downstream of TSSs. Sense A-repeats are enriched upstream, whereas sense T-repeats are enriched downstream of TSSs. This enrichment directly correlates with repeat size. Genes with different functions contain different lengths of repeats. In humans, tissue-specific genes are enriched for short repeats of <10 bp, whereas housekeeping genes are enriched for long repeats of ≥10 bp. We demonstrated that DICER1 and Argonaute proteins are required for the cis-regulatory role of A-repeats. Moreover, in the presence of a synthetic polymer that mimics an A-repeat, protein binding to A-repeats was blocked, resulting in a dramatic change in the expression of genes containing upstream A-repeats. Our findings suggest a length-dependent cis-regulatory function of A-repeats and that Argonaute proteins serve as trans-acting factors, binding to A-repeats.
DNA hybridization kinetics: zippering, internal displacement and sequence dependence [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Although the thermodynamics of DNA hybridization is generally well established, the kinetics of this classic transition is less well understood. Providing such understanding has new urgency because DNA nanotechnology often depends critically on binding rates. Here, we explore DNA oligomer hybridization kinetics using a coarse-grained model. Strand association proceeds through a complex set of intermediate states, with successful binding events initiated by a few metastable base-pairing interactions, followed by zippering of the remaining bonds. But despite reasonably strong interstrand interactions, initial contacts frequently dissociate because typical configurations in which they form differ from typical states of similar enthalpy in the double-stranded equilibrium ensemble. Initial contacts must be stabilized by two or three base pairs before full zippering is likely, resulting in negative effective activation enthalpies. Non-Arrhenius behavior arises because the number of base pairs required for nucleation increases with temperature. In addition, we observe two alternative pathways—pseudoknot and inchworm internal displacement—through which misaligned duplexes can rearrange to form duplexes. These pathways accelerate hybridization. Our results explain why experimentally observed association rates of GC-rich oligomers are higher than rates of AT- rich equivalents, and more generally demonstrate how association rates can be modulated by sequence choice.
Integrative analysis of SF-1 transcription factor dosage impact on genome-wide binding and gene expression regulation [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Steroidogenic Factor-1 (SF-1) is a nuclear receptor that has a pivotal role in the development of adrenal glands and gonads and in the control of steroid hormone production, being also implicated in the pathogenesis of adrenocortical tumors. We have analyzed the mechanisms how SF-1 controls gene expression in adrenocortical cells and showed that it regulates different categories of genes according to its dosage. Significant correlations exist between the localization of SF-1-binding sites in chromatin under different dosage conditions and dosage-dependent regulation of gene expression. Our study revealed unexpected functional interactions between SF-1 and Neuron-Restrictive Silencer Factor/RE1-Silencing Transcription Factor (NRSF/REST), which was first characterized as a repressor of neuronal gene expression in non-neuronal tissues, in the regulation of gene expression in steroidogenic cells. When overexpressed, SF-1 reshapes the repertoire of NRSF/REST—regulated genes, relieving repression of key steroidogenic genes. These data show that NRSF/REST has a novel function in regulating gene expression in steroidogenic cells and suggest that it may have a broad role in regulating tissue-specific gene expression programs.
Chromatin loop organization of the junb locus in mouse dendritic cells [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The junb gene behaves as an immediate early gene in bacterial lipopolysaccharide (LPS)-stimulated dendritic cells (DCs), where its transient transcriptional activation is necessary for the induction of inflammatory cytokines. junb is a short gene and its transcriptional activation by LPS depends on the binding of NF-B to an enhancer located just downstream of its 3' UTR. Here, we have addressed the mechanisms underlying the transcriptional hyper-reactivity of junb. Using transfection and pharmacological assays to complement chromatin immunoprecipitation analyses addressing the localization of histones, polymerase II, negative elongation factor (NELF)-, DRB sensitivity-inducing factor (DSIF)- and Positive Transcription Factor b complexes, we demonstrate that junb is a RNA Pol II-paused gene where Pol II is loaded in the transcription start site domain but poorly active. Moreover, High salt-Recovered Sequence, chromosome conformation capture (3C)- and gene transfer experiments show that (i) junb is organized in a nuclear chromatin loop bringing into close spatial proximity the upstream promoter region and the downstream enhancer and (ii) this configuration permits immediate Pol II release on the junb body on binding of LPS-activated NF-B to the enhancer. Thus, our work unveils a novel topological framework underlying fast junb transcriptional response in DCs. Moreover, it also points to a novel layer of complexity in the modes of action of NF-B.
A Common Docking Domain in Progesterone Receptor-B links DUSP6 and CK2 signaling to proliferative transcriptional programs in breast cancer cells [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Progesterone receptors (PR) are transcription factors relevant to breast cancer biology. Herein, we describe an N-terminal common docking (CD) domain in PR-B, a motif first described in mitogen-activated protein kinases. Binding studies revealed PR-B interacts with dual-specificity phosphatase 6 (DUSP6) via the CD domain. Mutation of the PR-B CD domain (mCD) attenuated cell cycle progression and expression of PR-B target genes (including STAT5A and Wnt1); mCD PR-B failed to undergo phosphorylation on Ser81, a ck2-dependent site required for expression of these genes. PR-B Ser81 phosphorylation was dependent on binding with DUSP6 and required for recruitment of a transcriptional complex consisting of PR-B, DUSP6 and ck2 to an enhancer region upstream of the Wnt1 promoter. STAT5 was present at this site in the absence or presence of progestin. Furthermore, phospho-Ser81 PR-B was recruited to the STAT5A gene upon progestin treatment, suggestive of a feed-forward mechanism. Inhibition of JAK/STAT-signaling blocked progestin-induced STAT5A and Wnt1 expression. Our studies show that DUSP6 serves as a scaffold for ck2-dependent PR-B Ser81 phosphorylation and subsequent PR-B-specific gene selection in coordination with STAT5. Coregulation of select target genes by PR-B and STAT5 is likely a global mechanism required for growth promoting programs relevant to mammary stem cell biology and cancer.
Temporal regulation of the Mus81-Mms4 endonuclease ensures cell survival under conditions of DNA damage [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The structure-specific Mus81-Eme1/Mms4 endonuclease contributes importantly to DNA repair and genome integrity maintenance. Here, using budding yeast, we have studied its function and regulation during the cellular response to DNA damage and show that this endonuclease is necessary for successful chromosome replication and cell survival in the presence of DNA lesions that interfere with replication fork progression. On the contrary, Mus81-Mms4 is not required for coping with replicative stress originated by acute treatment with hydroxyurea (HU), which causes fork stalling. Despite its requirement for dealing with DNA lesions that hinder DNA replication, Mus81-Mms4 activation is not induced by DNA damage at replication forks. Full Mus81-Mms4 activity is only acquired when cells finish S-phase and the endonuclease executes its function after the bulk of genome replication is completed. This post-replicative mode of action of Mus81-Mms4 limits its nucleolytic activity during S-phase, thus avoiding the potential cleavage of DNA substrates that could cause genomic instability during DNA replication. At the same time, it constitutes an efficient fail-safe mechanism for processing DNA intermediates that cannot be resolved by other proteins and persist after bulk DNA synthesis, which guarantees the completion of DNA repair and faithful chromosome replication when the DNA is damaged.
Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III {alpha} binding to ssDNA [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.
Many disease-associated variants of hTERT retain high telomerase enzymatic activity [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays.
XPD-dependent activation of apoptosis in response to triplex-induced DNA damage [Nucleic Acids Research - recent issues] 2013-10-18 05:10
DNA sequences capable of forming triplexes are prevalent in the human genome and have been found to be intrinsically mutagenic. Consequently, a balance between DNA repair and apoptosis is critical to counteract their effect on genomic integrity. Using triplex-forming oligonucleotides to synthetically create altered helical distortions, we have determined that pro-apoptotic pathways are activated by the formation of triplex structures. Moreover, the TFIIH factor, XPD, occupies a central role in triggering apoptosis in response to triplex-induced DNA strand breaks. Here, we show that triplexes are capable of inducing XPD-independent double strand breaks, which result in the formation of H2AX foci. XPD was subsequently recruited to the triplex-induced double strand breaks and co-localized with H2AX at the damage site. Furthermore, phosphorylation of H2AX tyrosine 142 was found to stimulate the signaling pathway of XPD-dependent apoptosis. We suggest that this mechanism may play an active role in minimizing genomic instability induced by naturally occurring noncanonical structures, perhaps protecting against cancer initiation.
Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Localized hyper-mutability caused by accumulation of lesions in persistent single-stranded (ss) DNA has been recently found in several types of cancers. An increase in endogenous levels of reactive oxygen species (ROS) is considered to be one of the hallmarks of cancers. Employing a yeast model system, we addressed the role of oxidative stress as a potential source of hyper-mutability in ssDNA by modulation of the endogenous ROS levels and by exposing cells to oxidative DNA-damaging agents. We report here that under oxidative stress conditions the majority of base substitution mutations in ssDNA are caused by erroneous, DNA polymerase (Pol) zeta-independent bypass of cytosines, resulting in C to T transitions. For all other DNA bases Pol zeta is essential for ROS-induced mutagenesis. The density of ROS-induced mutations in ssDNA is lower, compared to that caused by UV and MMS, which suggests that ssDNA could be actively protected from oxidative damage. These findings have important implications for understanding mechanisms of oxidative mutagenesis, and could be applied to development of anticancer therapies and cancer prevention.
Histone variant Htz1 promotes histone H3 acetylation to enhance nucleotide excision repair in Htz1 nucleosomes [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Nucleotide excision repair (NER) is critical for maintaining genome integrity. How chromatin dynamics are regulated to facilitate this process in chromatin is still under exploration. We show here that a histone H2A variant, Htz1 (H2A.Z), in nucleosomes has a positive function in promoting efficient NER in yeast. Htz1 inherently enhances the occupancy of the histone acetyltransferase Gcn5 on chromatin to promote histone H3 acetylation after UV irradiation. Consequently, this results in an increased binding of a NER protein, Rad14, to damaged DNA. Cells without Htz1 show increased UV sensitivity and defective removal of UV-induced DNA damage in the Htz1-bearing nucleosomes at the repressed MFA2 promoter, but not in the HMRa locus where Htz1 is normally absent. Thus, the effect of Htz1 on NER is specifically relevant to its presence in chromatin within a damaged region. The chromatin accessibility to micrococcal nuclease in the MFA2 promoter is unaffected by HTZ1 deletion. Acetylation on previously identified lysines of Htz1 plays little role in NER or cell survival after UV. In summary, we have identified a novel aspect of chromatin that regulates efficient NER, and we provide a model for how Htz1 influences NER in Htz1 nucleosomes.
Contributions of the RAD51 N-terminal domain to BRCA2-RAD51 interaction [Nucleic Acids Research - recent issues] 2013-10-18 05:10
RAD51 DNA strand exchange protein catalyzes the central step in homologous recombination, a cellular process fundamentally important for accurate repair of damaged chromosomes, preservation of the genetic integrity, restart of collapsed replication forks and telomere maintenance. BRCA2 protein, a product of the breast cancer susceptibility gene, is a key recombination mediator that interacts with RAD51 and facilitates RAD51 nucleoprotein filament formation on single-stranded DNA generated at the sites of DNA damage. An accurate atomistic level description of this interaction, however, is limited to a partial crystal structure of the RAD51 core fused to BRC4 peptide. Here, by integrating homology modeling and molecular dynamics, we generated a structure of the full-length RAD51 in complex with BRC4 peptide. Our model predicted previously unknown hydrogen bonding patterns involving the N-terminal domain (NTD) of RAD51. These interactions guide positioning of the BRC4 peptide within a cavity between the core and the NTDs; the peptide binding separates the two domains and restricts internal dynamics of RAD51 protomers. The model’s depiction of the RAD51-BRC4 complex was validated by free energy calculations and in vitro functional analysis of rationally designed mutants. All generated mutants, RAD51E42A, RAD51E59A, RAD51E237A, RAD51E59A/E237A and RAD51E42A/E59A/E237A maintained basic biochemical activities of the wild-type RAD51, but displayed reduced affinities for the BRC4 peptide. Strong correlation between the calculated and experimental binding energies confirmed the predicted structure of the RAD51-BRC4 complex and highlighted the importance of RAD51 NTD in RAD51-BRCA2 interaction.
High-resolution definition of the Vibrio cholerae essential gene set with hidden Markov model-based analyses of transposon-insertion sequencing data [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The coupling of high-density transposon mutagenesis to high-throughput DNA sequencing (transposon-insertion sequencing) enables simultaneous and genome-wide assessment of the contributions of individual loci to bacterial growth and survival. We have refined analysis of transposon-insertion sequencing data by normalizing for the effect of DNA replication on sequencing output and using a hidden Markov model (HMM)-based filter to exploit heretofore unappreciated information inherent in all transposon-insertion sequencing data sets. The HMM can smooth variations in read abundance and thereby reduce the effects of read noise, as well as permit fine scale mapping that is independent of genomic annotation and enable classification of loci into several functional categories (e.g. essential, domain essential or ‘sick’). We generated a high-resolution map of genomic loci (encompassing both intra- and intergenic sequences) that are required or beneficial for in vitro growth of the cholera pathogen, Vibrio cholerae. This work uncovered new metabolic and physiologic requirements for V. cholerae survival, and by combining transposon-insertion sequencing and transcriptomic data sets, we also identified several novel noncoding RNA species that contribute to V. cholerae growth. Our findings suggest that HMM-based approaches will enhance extraction of biological meaning from transposon-insertion sequencing genomic data.
Optimization of scarless human stem cell genome editing [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7–8x higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
Yeast Nop2 and Rcm1 methylate C2870 and C2278 of the 25S rRNA, respectively [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Yeast 25S rRNA was reported to contain a single cytosine methylation (m5C). In the present study using a combination of RP-HPLC, mung bean nuclease assay and rRNA mutagenesis, we discovered that instead of one, yeast contains two m5C residues at position 2278 and 2870. Furthermore, we identified and characterized two putative methyltransferases, Rcm1 and Nop2 to be responsible for these two cytosine methylations, respectively. Both proteins are highly conserved, which correlates with the presence of two m5C residues at identical positions in higher eukaryotes, including humans. The human homolog of yeast Nop2, p120 has been discovered to be upregulated in various cancer tissues, whereas the human homolog of Rcm1, NSUN5 is completely deleted in the William's-Beuren Syndrome. The substrates and function of both human homologs remained unknown. In the present study, we also provide insights into the significance of these two m5C residues. The loss of m5C2278 results in anisomycin hypersensitivity, whereas the loss of m5C2870 affects ribosome synthesis and processing. Establishing the locations and enzymes in yeast will not only help identifying the function of their homologs in higher organisms, but will also enable understanding the role of these modifications in ribosome function and architecture.
Alteration in the cavity size adjacent to the active site of RB69 DNA polymerase changes its conformational dynamics [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Internal cavities are a common feature of many proteins, often having profound effects on the dynamics of their interactions with substrate and binding partners. RB69 DNA polymerase (pol) has a hydrophobic cavity right below the nucleotide binding pocket at the tip of highly conserved L415 side chain. Replacement of this residue with Gly or Met in other B family pols resulted in higher mutation rates. When similar substitutions for L415 were introduced into RB69pol, only L415A and L415G had dramatic effects on pre-steady-state kinetic parameters, reducing base selectivity by several hundred fold. On the other hand, the L415M variant behaved like the wild-type. Using a novel tCo-tCnitro Förster Resonance Energy Transfer (FRET) assay, we were able to show that the partition of the primer terminus between pol and exonuclease (exo) domains was compromised with the L415A and L415G mutants, but not with the L415M variant. These results could be rationalized by changes in their structures as determined by high resolution X-ray crystallography.
Direct assessment of transcription fidelity by high-resolution RNA sequencing [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10–4–10–5 per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ~103 nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.
A specific N-terminal extension of the 8 kDa domain is required for DNA end-bridging by human Pol{micro} and Pol{lambda} [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Human DNA polymerases mu (Polµ) and lambda (Pol) are X family members involved in the repair of double-strand breaks in DNA during non-homologous end joining. Crucial abilities of these enzymes include bridging of the two 3' single-stranded overhangs and trans-polymerization using one 3' end as primer and the other as template, to minimize sequence loss. In this context, we have studied the importance of a previously uncharacterised sequence (‘brooch’), located at the N-terminal boundary of the Polß-like polymerase core, and formed by Tyr141, Ala142, Cys143, Gln144 and Arg145 in Polµ, and by Trp239, Val240, Cys241, Ala242 and Gln243 in Pol. The brooch is potentially implicated in the maintenance of a closed conformation throughout the catalytic cycle, and our studies indicate that it could be a target of Cdk phosphorylation in Polµ. The brooch is irrelevant for 1 nt gap filling, but of specific importance during end joining: single mutations in the conserved residues reduced the formation of two ended synapses and strongly diminished the ability of Polµ and polymerase lambda to perform non-homologous end joining reactions in vitro.
A three-state model for the regulation of telomerase by TERRA and hnRNPA1 [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Telomeres, the physical ends of eukaryotic chromosomes, are transcribed into telomeric repeat-containing RNA (TERRA), a large non-coding RNA, which forms an integral part of telomeric heterochromatin. In vitro, naked TERRA molecules are efficient inhibitors of human telomerase, base-pairing via their 5'-UUAGGG-3' repeats with the template sequence of telomerase RNA, in addition to contacting the telomerase reverse transcriptase protein subunit. In vivo, however, TERRA-mediated inhibition of telomerase can be prevented by unknown mechanisms. Also, heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has been implicated in telomere length control. In vivo, TERRA is partially associated with hnRNPA1, and hnRNPA1 is also detected at telomeres. We demonstrate that on binding of TERRA, hnRNPA1 can alleviate the TERRA-mediated inhibition of telomerase. However, when in excess over TERRA, hnRNPA1 becomes itself an inhibitor of telomere extension, on binding of the telomeric DNA substrate. Yet, hnRNPA1 has no notable direct effects on the telomerase catalysis. Our in vitro results suggest that TERRA-mediated telomerase inhibition may be prevented by hnRNPA1 in vivo. Telomere extension by telomerase may require balanced levels of TERRA and hnRNPA1 at telomeres. Thus, TERRA and hnRNPA1 can function as a bimolecular regulator to turn telomerase and the telomere on and off.
Dissecting the interactions of SERRATE with RNA and DICER-LIKE 1 in Arabidopsis microRNA precursor processing [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Efficient and precise microRNA (miRNA) biogenesis in Arabidopsis is mediated by the RNaseIII-family enzyme DICER-LIKE 1 (DCL1), double-stranded RNA-binding protein HYPONASTIC LEAVES 1 and the zinc-finger (ZnF) domain-containing protein SERRATE (SE). In the present study, we examined primary miRNA precursor (pri-miRNA) processing by highly purified recombinant DCL1 and SE proteins and found that SE is integral to pri-miRNA processing by DCL1. SE stimulates DCL1 cleavage of the pri-miRNA in an ionic strength-dependent manner. SE uses its N-terminal domain to bind to RNA and requires both N-terminal and ZnF domains to bind to DCL1. However, when DCL1 is bound to RNA, the interaction with the ZnF domain of SE becomes indispensible and stimulates the activity of DCL1 without requiring SE binding to RNA. Our results suggest that the interactions among SE, DCL1 and RNA are a potential point for regulating pri-miRNA processing.
RNase J participates in a pentatricopeptide repeat protein-mediated 5' end maturation of chloroplast mRNAs [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Nucleus-encoded ribonucleases and RNA-binding proteins influence chloroplast gene expression through their roles in RNA maturation and stability. One mechanism for mRNA 5' end maturation posits that sequence-specific pentatricopeptide repeat (PPR) proteins define termini by blocking the 5'->3' exonucleolytic activity of ribonuclease J (RNase J). To test this hypothesis in vivo, virus-induced gene silencing was used to reduce the expression of three PPR proteins and RNase J, both individually and jointly, in Nicotiana benthamiana. In accordance with the stability-conferring function of the PPR proteins PPR10, HCF152 and MRL1, accumulation of the cognate RNA species atpH, petB and rbcL was reduced when the PPR-encoding genes were silenced. In contrast, RNase J reduction alone or combined with PPR deficiency resulted in reduced abundance of polycistronic precursor transcripts and mature counterparts, which were replaced by intermediately sized species with heterogeneous 5' ends. We conclude that RNase J deficiency can partially mask the absence of PPR proteins, and that RNase J is capable of processing chloroplast mRNAs up to PPR protein-binding sites. These findings support the hypothesis that RNase J is the major ribonuclease responsible for maturing chloroplast mRNA 5' termini, with RNA-binding proteins acting as barriers to its activity.
RNA-dependent association with myosin IIA promotes F-actin-guided trafficking of the ELAV-like protein HuR to polysomes [Nucleic Acids Research - recent issues] 2013-10-18 05:10
The role of the mRNA-binding protein human antigen R (HuR) in stabilization and translation of AU-rich elements (ARE) containing mRNAs is well established. However, the trafficking of HuR and bound mRNA cargo, which comprises a fundamental requirement for the aforementioned HuR functions is only poorly understood. By administering different cytoskeletal inhibitors, we found that the protein kinase C (PKC)-triggered accumulation of cytoplasmic HuR by Angiotensin II (AngII) is an actin-myosin driven process functionally relevant for stabilization of ARE-bearing mRNAs. Furthermore, we show that the AngII-induced recruitment of HuR and its bound mRNA from ribonucleoprotein particles to free and cytoskeleton bound polysomes strongly depended on an intact actomyosin cytoskeleton. In addition, HuR allocation to free and cytoskeletal bound polysomes is highly sensitive toward RNase and PPtase and structurally depends on serine 318 (S318) located within the C-terminal RNA recognition motif (RRM3). Conversely, the trafficking of the phosphomimetic HuRS318D, mimicking HuR phosphorylation at S318 by the PKC remained PPtase resistant. Co-immunoprecipitation experiments with truncated HuR proteins revealed that the stimulus-induced association of HuR with myosin IIA is strictly RNA dependent and mediated via the RRM3. Our data implicate a microfilament dependent transport of HuR, which is relevant for stimulus-induced targeting of ARE-bearing mRNAs from translational inactive ribonucleoprotein particles to polysomes.
The BAH domain of Rsc2 is a histone H3 binding domain [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction.
RNA polymerase III-specific general transcription factor IIIC contains a heterodimer resembling TFIIF Rap30/Rap74 [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Transcription of tRNA-encoding genes by RNA polymerase (Pol) III requires the six-subunit general transcription factor IIIC that uses subcomplexes A and B to recognize two gene-internal promoter elements named A- and B-box. The Schizosaccharomyces pombe A subcomplex comprises subunits Sfc1, Sfc4 and Sfc7. The crystal structure of the Sfc1/Sfc7 heterodimer reveals similar domains and overall domain architecture to the Pol II-specific general transcription factor TFIIF Rap30/Rap74. The N-terminal Sfc1/Sfc7 dimerization module consists of a triple β-barrel similar to the N-terminal TFIIF Rap30/Rap74 dimerization module, whereas the C-terminal Sfc1 DNA-binding domain contains a winged-helix domain most similar to the TFIIF Rap30 C-terminal winged-helix domain. Sfc1 DNA-binding domain recognizes single and double-stranded DNA by an unknown mechanism. Several features observed for A-box recognition by A resemble the recognition of promoters by bacterial RNA polymerase, where factor unfolds double-stranded DNA and stabilizes the non-coding DNA strand in an open conformation. Such a function has also been proposed for TFIIF, suggesting that the observed structural similarity between Sfc1/Sfc7 and TFIIF Rap30/Rap74 might also reflect similar functions.
Transcription activator like effector (TALE)-directed piggyBac transposition in human cells [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations.
NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations [Nucleic Acids Research - recent issues] 2013-10-18 05:10
Nucleic Acids Research: VOLUME 41 ISSUE 18 2013 [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Nucleic Acids Research - Editorial Board [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Subscriptions [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Enrichment analysis of gene sets is a popular approach that provides a functional interpretation of genome-wide expression data. Existing tests are affected by inter-gene correlations, resulting in a high Type I error. The most widely used test, Gene Set Enrichment Analysis, relies on computationally intensive permutations of sample labels to generate a null distribution that preserves gene–gene correlations. A more recent approach, CAMERA, attempts to correct for these correlations by estimating a variance inflation factor directly from the data. Although these methods generate P-values for detecting gene set activity, they are unable to produce confidence intervals or allow for post hoc comparisons. We have developed a new computational framework for Quantitative Set Analysis of Gene Expression (QuSAGE). QuSAGE accounts for inter-gene correlations, improves the estimation of the variance inflation factor and, rather than evaluating the deviation from a null hypothesis with a P-value, it quantifies gene-set activity with a complete probability density function. From this probability density function, P-values and confidence intervals can be extracted and post hoc analysis can be carried out while maintaining statistical traceability. Compared with Gene Set Enrichment Analysis and CAMERA, QuSAGE exhibits better sensitivity and specificity on real data profiling the response to interferon therapy (in chronic Hepatitis C virus patients) and Influenza A virus infection. QuSAGE is available as an R package, which includes the core functions for the method as well as functions to plot and visualize the results.
Concordance of gene expression in human protein complexes reveals tissue specificity and pathology [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Disease-causing variants in human genes usually lead to phenotypes specific to only a few tissues. Here, we present a method for predicting tissue specificity based on quantitative deregulation of protein complexes. The underlying assumption is that the degree of coordinated expression among proteins in a complex within a given tissue may pinpoint tissues that will be affected by a mutation in the complex and coordinated expression may reveal the complex to be active in the tissue. We identified known disease genes and their protein complex partners in a high-quality human interactome. Each susceptibility gene's tissue involvement was ranked based on coordinated expression with its interaction partners in a non-disease global map of human tissue-specific expression. The approach demonstrated high overall area under the curve (0.78) and was very successfully benchmarked against a random model and an approach not using protein complexes. This was illustrated by correct tissue predictions for three case studies on leptin, insulin-like-growth-factor 2 and the inhibitor of NF-B kinase subunit gamma that show high concordant expression in biologically relevant tissues. Our method identifies novel gene-phenotype associations in human diseases and predicts the tissues where associated phenotypic effects may arise.
Automated and assisted RNA resonance assignment using NMR chemical shift statistics [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The three-dimensional structure determination of RNAs by NMR spectroscopy relies on chemical shift assignment, which still constitutes a bottleneck. In order to develop more efficient assignment strategies, we analysed relationships between sequence and 1H and 13C chemical shifts. Statistics of resonances from regularly Watson–Crick base-paired RNA revealed highly characteristic chemical shift clusters. We developed two approaches using these statistics for chemical shift assignment of double-stranded RNA (dsRNA): a manual approach that yields starting points for resonance assignment and simplifies decision trees and an automated approach based on the recently introduced automated resonance assignment algorithm FLYA. Both strategies require only unlabeled RNAs and three 2D spectra for assigning the H2/C2, H5/C5, H6/C6, H8/C8 and H1'/C1' chemical shifts. The manual approach proved to be efficient and robust when applied to the experimental data of RNAs with a size between 20 nt and 42 nt. The more advanced automated assignment approach was successfully applied to four stem-loop RNAs and a 42 nt siRNA, assigning 92–100% of the resonances from dsRNA regions correctly. This is the first automated approach for chemical shift assignment of non-exchangeable protons of RNA and their corresponding 13C resonances, which provides an important step toward automated structure determination of RNAs.
Targeted resequencing of HIV variants by microarray thermodynamics [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20–30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes.
In-gel probing of individual RNA conformers within a mixed population reveals a dimerization structural switch in the HIV-1 leader [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Definitive secondary structural mapping of RNAs in vitro can be complicated by the presence of more than one structural conformer or multimerization of some of the molecules. Until now, probing a single structure of conformationally flexible RNA molecules has typically relied on introducing stabilizing mutations or adjusting buffer conditions or RNA concentration. Here, we present an in-gel SHAPE (selective 2'OH acylation analysed by primer extension) approach, where a mixed structural population of RNA molecules is separated by non-denaturing gel electrophoresis and the conformers are individually probed within the gel matrix. Validation of the technique using a well-characterized RNA stem-loop structure, the HIV-1 trans-activation response element, showed that authentic structure was maintained and that the method was accurate and highly reproducible. To further demonstrate the utility of in-gel SHAPE, we separated and examined monomeric and dimeric species of the HIV-1 packaging signal RNA. Extensive differences in acylation sensitivity were seen between monomer and dimer. The results support a recently proposed structural switch model of RNA genomic dimerization and packaging, and demonstrate the discriminatory power of in-gel SHAPE.
Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.
In situ visualization of telomere elongation patterns in human cells [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The telomerase enzyme plays a critical role in human aging and cancer biology by maintaining telomere length and extending the proliferative lifespan of most stem cells and cancer cells. Despite the importance of this enzyme, our understanding of the mechanisms that regulate its activity and establish telomere length homeostasis in mammalian cells is incomplete, in part because the perfect repetitive nature of telomeric sequence hampers in situ detection of telomere elongation patterns. Here, we describe a novel assay using a mutant telomerase that adds a well-tolerated variant telomeric repeat sequence to telomere ends. By specifically detecting the addition of these variant repeats, we can directly visualize telomere elongation events in human cells. We validate this approach by in situ mapping of telomere elongation patterns within individual nuclei and across a population of cells.
Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNAIleUAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.
Transcriptional regulation of human DNA repair genes following genotoxic stress: trigger mechanisms, inducible responses and genotoxic adaptation [Nucleic Acids Research - recent issues] 2013-10-07 01:00
DNA repair is the first barrier in the defense against genotoxic stress. In recent years, mechanisms that recognize DNA damage and activate DNA repair functions through transcriptional upregulation and post-translational modification were the focus of intensive research. Most DNA repair pathways are complex, involving many proteins working in discrete consecutive steps. Therefore, their balanced expression is important for avoiding erroneous repair that might result from excessive base removal and DNA cleavage. Amelioration of DNA repair requires both a fine-tuned system of lesion recognition and transcription factors that regulate repair genes in a balanced way. Transcriptional upregulation of DNA repair genes by genotoxic stress is counteracted by DNA damage that blocks transcription. Therefore, induction of DNA repair resulting in an adaptive response is only visible through a narrow window of dose. Here, we review transcriptional regulation of DNA repair genes in normal and cancer cells and describe mechanisms of promoter activation following genotoxic exposures through environmental carcinogens and anticancer drugs. The data available to date indicate that 25 DNA repair genes are subject to regulation following genotoxic stress in rodent and human cells, but for only a few of them, the data are solid as to the mechanism, homeostatic regulation and involvement in an adaptive response to genotoxic stress.
MREdictor: a two-step dynamic interaction model that accounts for mRNA accessibility and Pumilio binding accurately predicts microRNA targets [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The prediction of pairing between microRNAs (miRNAs) and the miRNA recognition elements (MREs) on mRNAs is expected to be an important tool for understanding gene regulation. Here, we show that mRNAs that contain Pumilio recognition elements (PRE) in the proximity of predicted miRNA-binding sites are more likely to form stable secondary structures within their 3'-UTR, and we demonstrated using a PUM1 and PUM2 double knockdown that Pumilio proteins are general regulators of miRNA accessibility. On the basis of these findings, we developed a computational method for predicting miRNA targets that accounts for the presence of PRE in the proximity of seed-match sequences within poorly accessible structures. Moreover, we implement the miRNA-MRE duplex pairing as a two-step model, which better fits the available structural data. This algorithm, called MREdictor, allows for the identification of miRNA targets in poorly accessible regions and is not restricted to a perfect seed-match; these features are not present in other computational prediction methods.
Evidence of direct complementary interactions between messenger RNAs and their cognate proteins [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Recently, the ability to interact with messenger RNA (mRNA) has been reported for a number of known RNA-binding proteins, but surprisingly also for different proteins without recognizable RNA binding domains including several transcription factors and metabolic enzymes. Moreover, direct binding to cognate mRNAs has been detected for multiple proteins, thus creating a strong impetus to search for functional significance and basic physico-chemical principles behind such interactions. Here, we derive interaction preferences between amino acids and RNA bases by analyzing binding interfaces in the known 3D structures of protein–RNA complexes. By applying this tool to human proteome, we reveal statistically significant matching between the composition of mRNA sequences and base-binding preferences of protein sequences they code for. For example, purine density profiles of mRNA sequences mirror guanine affinity profiles of cognate protein sequences with quantitative accuracy (median Pearson correlation coefficient R = –0.80 across the entire human proteome). Notably, statistically significant anti-matching is seen only in the case of adenine. Our results provide strong evidence for the stereo-chemical foundation of the genetic code and suggest that mRNAs and cognate proteins may in general be directly complementary to each other and associate, especially if unstructured.
Minimal genome encoding proteins with constrained amino acid repertoire [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Minimal bacterial gene set comprises the genetic elements needed for survival of engineered bacterium on a rich medium. This set is estimated to include 300–350 protein-coding genes. One way of simplifying an organism with such a minimal genome even further is to constrain the amino acid content of its proteins. In this study, comparative genomics approaches and the results of gene knockout experiments were used to extrapolate the minimal gene set of mollicutes, and bioinformatics combined with the knowledge-based analysis of the structure-function relationships in these proteins and their orthologs, paralogs and analogs was applied to examine the challenges of completely replacing the rarest residue, cysteine. Among several known functions of cysteine residues, their roles in the active centers of the enzymes responsible for deoxyribonucleoside synthesis and transfer RNA modification appear to be crucial, as no alternative chemistry is known for these reactions. Thus, drastic reduction of the content of the rarest amino acid in a minimal proteome appears to be possible, but its complete elimination is challenging.
A comprehensive gene regulatory network for the diauxic shift in Saccharomyces cerevisiae [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Existing machine-readable resources for large-scale gene regulatory networks usually do not provide context information characterizing the activating conditions for a regulation and how targeted genes are affected. Although this information is essentially required for data interpretation, available networks are often restricted to not condition-dependent, non-quantitative, plain binary interactions as derived from high-throughput screens. In this article, we present a comprehensive Petri net based regulatory network that controls the diauxic shift in Saccharomyces cerevisiae. For 100 specific enzymatic genes, we collected regulations from public databases as well as identified and manually curated >400 relevant scientific articles. The resulting network consists of >300 multi-input regulatory interactions providing (i) activating conditions for the regulators; (ii) semi-quantitative effects on their targets; and (iii) classification of the experimental evidence. The diauxic shift network compiles widespread distributed regulatory information and is available in an easy-to-use machine-readable form. Additionally, we developed a browsable system organizing the network into pathway maps, which allows to inspect and trace the evidence for each annotated regulation in the model.
Integrated analysis of genome-wide DNA methylation and gene expression profiles in molecular subtypes of breast cancer [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Aberrant DNA methylation of CpG islands, CpG island shores and first exons is known to play a key role in the altered gene expression patterns in all human cancers. To date, a systematic study on the effect of DNA methylation on gene expression using high resolution data has not been reported. In this study, we conducted an integrated analysis of MethylCap-sequencing data and Affymetrix gene expression microarray data for 30 breast cancer cell lines representing different breast tumor phenotypes. As well-developed methods for the integrated analysis do not currently exist, we created a series of four different analysis methods. On the computational side, our goal is to develop methylome data analysis protocols for the integrated analysis of DNA methylation and gene expression data on the genome scale. On the cancer biology side, we present comprehensive genome-wide methylome analysis results for differentially methylated regions and their potential effect on gene expression in 30 breast cancer cell lines representing three molecular phenotypes, luminal, basal A and basal B. Our integrated analysis demonstrates that methylation status of different genomic regions may play a key role in establishing transcriptional patterns in molecular subtypes of human breast cancer.
Analysis of epigenetic stability and conversions in Saccharomyces cerevisiae reveals a novel role of CAF-I in position-effect variegation [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Position-effect variegation (PEV) phenotypes are characterized by the robust multigenerational repression of a gene located at a certain locus (often called gene silencing) and occasional conversions to fully active state. Consequently, the active state then persists with occasional conversions to the repressed state. These effects are mediated by the establishment and maintenance of heterochromatin or euchromatin structures, respectively. In this study, we have addressed an important but often neglected aspect of PEV: the frequency of conversions at such loci. We have developed a model and have projected various PEV scenarios based on various rates of conversions. We have also enhanced two existing assays for gene silencing in Saccharomyces cerevisiae to measure the rate of switches from repressed to active state and vice versa. We tested the validity of our methodology in sir1 cells and in several mutants with defects in gene silencing. The assays have revealed that the histone chaperone Chromatin Assembly Factor I is involved in the control of epigenetic conversions. Together, our model and assays provide a comprehensive methodology for further investigation of epigenetic stability and position effects.
The PRP6-like splicing factor STA1 is involved in RNA-directed DNA methylation by facilitating the production of Pol V-dependent scaffold RNAs [Nucleic Acids Research - recent issues] 2013-10-07 01:00
DNA methylation is a conserved epigenetic marker in plants and animals. In Arabidopsis, DNA methylation can be established through an RNA-directed DNA methylation (RdDM) pathway. By screening for suppressors of ros1, we identified STA1, a PRP6-like splicing factor, as a new RdDM regulator. Whole-genome bisulfite sequencing suggested that STA1 and the RdDM pathway share a large number of common targets in the Arabidopsis genome. Small RNA deep sequencing demonstrated that STA1 is predominantly involved in the accumulation of the siRNAs that depend on both Pol IV and Pol V. Moreover, the sta1 mutation partially reduces the levels of Pol V-dependent RNA transcripts. Immunolocalization assay indicated that STA1 signals are exclusively present in the Cajal body and overlap with AGO4 in most nuclei. STA1 signals are also partially overlap with NRPE1. Localization of STA1 to AGO4 and NRPE1 signals is probably related to the function of STA1 in the RdDM pathway. Based on these results, we propose that STA1 acts downstream of siRNA biogenesis and facilitates the production of Pol V-dependent RNA transcripts in the RdDM pathway.
Integrative analysis of tissue-specific methylation and alternative splicing identifies conserved transcription factor binding motifs [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The exact role of intragenic DNA methylation in regulating tissue-specific gene regulation is unclear. Recently, the DNA-binding protein CTCF has been shown to participate in the regulation of alternative splicing in a DNA methylation-dependent manner. To globally evaluate the relationship between DNA methylation and tissue-specific alternative splicing, we performed genome-wide DNA methylation profiling of mouse retina and brain. In protein-coding genes, tissue-specific differentially methylated regions (T-DMRs) were preferentially located in exons and introns. Gene ontology and evolutionary conservation analysis suggest that these T-DMRs are likely to be biologically relevant. More than 14% of alternatively spliced genes were associated with a T-DMR. T-DMR-associated genes were enriched for developmental genes, suggesting that a specific set of alternatively spliced genes may be regulated through DNA methylation. Novel DNA sequences motifs overrepresented in T-DMRs were identified as being associated with positive and/or negative regulation of alternative splicing in a position-dependent context. The majority of these evolutionarily conserved motifs contain a CpG dinucleotide. Some transcription factors, which recognize these motifs, are known to be involved in splicing. Our results suggest that DNA methylation-dependent alternative splicing is widespread and lay the foundation for further mechanistic studies of the role of DNA methylation in tissue-specific splicing regulation.
Pin1 promotes GR transactivation by enhancing recruitment to target genes [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The glucocorticoid receptor (GR) is a ligand activated transcription factor, serving to regulate both energy metabolism and immune functions. Factors that influence cellular sensitivity to glucocorticoids (GC) are therefore of great interest. The N-terminal of the GR contains numerous potential proline-directed phosphorylation sites, some of which can regulate GR transactivation. Unrestricted proline isomerisation can be inhibited by adjacent serine phosphorylation and requires a prolyl isomerise, Pin1. Pin1 therefore determines the functional outcome of proline-directed kinases acting on the GR, as cis/trans isomers are distinct pools with different interacting proteins. We show that Pin1 mediates GR transactivation, but not GR trans-repression. Two N-terminal GR serines, S203 and S211, are targets for Pin1 potentiation of GR transactivation, establishing a direct link between Pin1 and the GR. We also demonstrate GC-activated co-recruitment of GR and Pin1 to the GILZ gene promoter. The Pin1 effect required both its WW and catalytic domains, and GR recruitment to its GRE was Pin1-dependent. Therefore, Pin1 is a selective regulator of GR transactivation, acting through N-terminal phospho-serine residues to regulate GR recruitment to its target sites in the genome. As Pin1 is dysregulated in disease states, this interaction may contribute to altered GC action in inflammatory conditions.
CCAR1 promotes chromatin loading of androgen receptor (AR) transcription complex by stabilizing the association between AR and GATA2 [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Androgen receptor (AR), a ligand-dependent transcription factor, plays a critical role in prostate cancer onset and progression, and its transcriptional function is mediated largely by distinct nuclear receptor co-regulators. Here, we show that cell cycle and apoptosis regulator 1 (CCAR1) functions as an AR co-activator. CCAR1 interacted with and enhanced the transcriptional activity of AR. Depletion of CCAR1 caused reduction in androgen-dependent expression of a subset of AR target genes. We further showed that CCAR1 is required for recruitment of AR, MED1 and RNA polymerase II to the enhancers of AR target genes and for androgen-induced long-range prostate specific antigen enhancer–promoter interaction. The molecular mechanism underlying CCAR1 function in AR-mediated transcription involves CCAR1-mediated enhanced recruitment of GATA2, a pioneer factor for AR, to AR-binding sites. CCAR1 stabilized the interaction between AR and GATA2 by interacting directly with both proteins, thereby facilitating AR and GATA2 occupancy on the enhancers. Furthermore, CCAR1 depletion inhibited the growth, migration, invasion of prostate cancer cells and reduced the tumorigenicity of prostate cancer cells in vivo. Our results firmly established CCAR1 as an AR co-activator that plays a key role in AR transcription complex assembly and has an important physiological role in androgen signaling and prostate tumorigenesis.
SIRT6 exhibits nucleosome-dependent deacetylase activity [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The SIRT6 deacetylase is a key regulator of mammalian genome stability, metabolism and lifespan. Previous studies indicated that SIRT6 exhibits poor deacetylase activity in vitro. Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase. We show that SIRT6 associates with the nucleosome and deacetylates histones H3 and H4 when they are packaged as nucleosomes, but not as free histones. In contrast, SIRT1 shows the opposite characteristics. Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.
Protein-DNA binding dynamics predict transcriptional response to nutrients in archaea [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Organisms across all three domains of life use gene regulatory networks (GRNs) to integrate varied stimuli into coherent transcriptional responses to environmental pressures. However, inferring GRN topology and regulatory causality remains a central challenge in systems biology. Previous work characterized TrmB as a global metabolic transcription factor in archaeal extremophiles. However, it remains unclear how TrmB dynamically regulates its ~100 metabolic enzyme-coding gene targets. Using a dynamic perturbation approach, we elucidate the topology of the TrmB metabolic GRN in the model archaeon Halobacterium salinarum. Clustering of dynamic gene expression patterns reveals that TrmB functions alone to regulate central metabolic enzyme-coding genes but cooperates with various regulators to control peripheral metabolic pathways. Using a dynamical model, we predict gene expression patterns for some TrmB-dependent promoters and infer secondary regulators for others. Our data suggest feed-forward gene regulatory topology for cobalamin biosynthesis. In contrast, purine biosynthesis appears to require TrmB-independent regulators. We conclude that TrmB is an important component for mediating metabolic modularity, integrating nutrient status and regulating gene expression dynamics alone and in concert with secondary regulators.
Modulation of base excision repair of 8-oxoguanine by the nucleotide sequence [Nucleic Acids Research - recent issues] 2013-10-07 01:00
8-Oxoguanine (8-oxoG) is a major product of oxidative DNA damage, which induces replication errors and interferes with transcription. By varying the position of single 8-oxoG in a functional gene and manipulating the nucleotide sequence surrounding the lesion, we found that the degree of transcriptional inhibition is independent of the distance from the transcription start or the localization within the transcribed or the non-transcribed DNA strand. However, it is strongly dependent on the sequence context and also proportional to cellular expression of 8-oxoguanine DNA glycosylase (OGG1)—demonstrating that transcriptional arrest does not take place at unrepaired 8-oxoG and proving a causal connection between 8-oxoG excision and the inhibition of transcription. We identified the 5'-CAGGGC[8-oxoG]GACTG-3' motif as having only minimal transcription-inhibitory potential in cells, based on which we predicted that 8-oxoG excision is particularly inefficient in this sequence context. This anticipation was fully confirmed by direct biochemical assays. Furthermore, in DNA containing a bistranded Cp[8-oxoG]/Cp[8-oxoG] clustered lesion, the excision rates differed between the two strands at least by a factor of 9, clearly demonstrating that the excision preference is defined by the DNA strand asymmetry rather than the overall geometry of the double helix or local duplex stability.
The ubiquitin specific protease USP34 promotes ubiquitin signaling at DNA double-strand breaks [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Ubiquitylation plays key roles in DNA damage signal transduction. The current model envisions that lysine63-linked ubiquitin chains, via the concerted action of E3 ubiquitin ligases RNF8-RNF168, are built at DNA double-strand breaks (DSBs) to effectively assemble DNA damage-repair factors for proper checkpoint control and DNA repair. We found that RNF168 is a short-lived protein that is stabilized by the deubiquitylating enzyme USP34 in response to DNA damage. In the absence of USP34, RNF168 is rapidly degraded, resulting in attenuated DSB-associated ubiquitylation, defective recruitment of BRCA1 and 53BP1 and compromised cell survival after ionizing radiation. We propose that USP34 promotes a feed-forward loop to enforce ubiquitin signaling at DSBs and highlight critical roles of ubiquitin dynamics in genome stability maintenance.
Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules [Nucleic Acids Research - recent issues] 2013-10-07 01:00
DNA lesions produced by aromatic isocyanates have an extra bulky group on the nucleotide bases, with the capability of forming stacking interaction within a DNA helix. In this work, we investigated the conformation of the 2'-deoxyadenosine and 2'-deoxycytidine derivatives tethering a phenyl or naphthyl group, introduced in a DNA duplex. The chemical modification experiments using KMnO4 and 1-cyclohexyl-3 -(2-morpholinoethyl) carbodiimide metho-p-toluenesulfonate have shown that the 2'-deoxycytidine lesions form the base pair with guanine while the 2'-deoxyadenosine lesions have less ability of forming the base pair with thymine in solution. Nevertheless, the kinetic analysis shows that these DNA lesions are compatible with DNA ligase and DNA polymerase reactions, as much as natural DNA bases. We suggest that the adduct lesions have a capability of adopting dual conformations, depending on the difference in their interaction energies between stacking of the attached aromatic group and base pairing through hydrogen bonds. It is also presented that the attached aromatic groups change their orientation by interacting with the minor groove binding netropsin, distamycin and synthetic polyamide. The nucleotide derivatives would be useful for enhancing the phenotypic diversity of DNA molecules and for exploring new non-natural nucleotides.
Global identification of conserved post-transcriptional regulatory programs in trypanosomatids [Nucleic Acids Research - recent issues] 2013-10-07 01:00
While regulatory programs are extensively studied at the level of transcription, elements that are involved in regulation of post-transcriptional processes are largely unknown, and methods for systematic identification of these elements are in early stages. Here, using a novel computational framework, we have integrated sequence information with several functional genomics data sets to characterize conserved regulatory programs of trypanosomatids, a group of eukaryotes that almost entirely rely on post-transcriptional processes for regulation of mRNA abundance. This analysis revealed a complex network of linear and structural RNA elements that potentially govern mRNA abundance across different life stages and environmental conditions. Furthermore, we show that the conserved regulatory network that we have identified is responsive to chemical perturbation of several biological functions in trypanosomatids. We have further characterized one of the most abundant regulatory RNA elements that we discovered, an AU-rich element (ARE) that can be found in 3' untranslated region of many trypanosomatid genes. Using bioinformatics approaches as well as in vitro and in vivo experiments, we have identified three ELAV-like homologs, including the developmentally critical protein TbRBP6, which regulate abundance of a large number of trypanosomatid ARE-containing transcripts. Together, these studies lay out a roadmap for characterization of mechanisms that modulate development and metabolic pathways in trypanosomatids.
BRCA1 is a key regulator of breast differentiation through activation of Notch signalling with implications for anti-endocrine treatment of breast cancers [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Here, we show for the first time, that the familial breast/ovarian cancer susceptibility gene BRCA1 activates the Notch pathway in breast cells by transcriptional upregulation of Notch ligands and receptors in both normal and cancer cells. We demonstrate through chromatin immunoprecipitation assays that BRCA1 is localized to a conserved intronic enhancer region within the Notch ligand Jagged-1 (JAG1) gene, an event requiring Np63. We propose that this BRCA1/Np63-mediated induction of JAG1 may be important the regulation of breast stem/precursor cells, as knockdown of all three proteins resulted in increased tumoursphere growth and increased activity of stem cell markers such as Aldehyde Dehydrogenase 1 (ALDH1). Knockdown of Notch1 and JAG1 phenocopied BRCA1 knockdown resulting in the loss of Estrogen Receptor-α (ER-α) expression and other luminal markers. A Notch mimetic peptide could activate an ER-α promoter reporter in a BRCA1-dependent manner, whereas Notch inhibition using a -secretase inhibitor reversed this process. We demonstrate that inhibition of Notch signalling resulted in decreased sensitivity to the anti-estrogen drug Tamoxifen but increased expression of markers associated with basal-like breast cancer. Together, these findings suggest that BRCA1 transcriptional upregulation of Notch signalling is a key event in the normal differentiation process in breast tissue.
Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA) [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNAAsp(GUC) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNAGlu(CUC/UUC) and tRNAGly(GCC) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.
Carboxy terminal modifications of the P0 protein reveal alternative mechanisms of nuclear ribosomal stalk assembly [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The P0 scaffold protein of the ribosomal stalk is mainly incorporated into pre-ribosomes in the cytoplasm where it replaces the assembly factor Mrt4. In analyzing the role of the P0 carboxyl terminal domain (CTD) during ribosomal stalk assembly, we found that its complete removal yields a protein that is functionally similar to Mrt4, whereas a chimeric Mrt4 containing the P0 CTD behaves more like P0. Deleting the P0 binding sites for the P1 and P2 proteins provoked the nuclear accumulation of P0AB induced by either leptomycin B-mediated blockage of nuclear export or Mrt4 deletion. This effect was reversed by removing P1/P2 from the cell, whereas nuclear accumulation was restored on reintroduction of these proteins. Together, these results indicate that the CTD determines the function of the P0 in stalk assembly. Moreover, they indicate that in cells lacking Mrt4, P0 and its stalk base partner, the L12 protein, bind to pre-ribosomes in the nucleus, a complex that is then exported to the cytoplasm by a mechanism assisted by the interaction with P1/P2 proteins. Furthermore, in wild-type cells, the presence of nuclear pre-ribosome complexes containing P0 but not L12 is compatible with the existence of an alternative stalk assembly process.
Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.
Early steps of active DNA demethylation initiated by ROS1 glycosylase require three putative helix-invading residues [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Active DNA demethylation is crucial for epigenetic control, but the underlying enzymatic mechanisms are incompletely understood. REPRESSOR OF SILENCING 1 (ROS1) is a 5-methylcytosine (5-meC) DNA glycosylase/lyase that initiates DNA demethylation in plants through a base excision repair process. The enzyme binds DNA nonspecifically and slides along the substrate in search of 5-meC. In this work, we have used homology modelling and biochemical analysis to gain insight into the mechanism of target location and recognition by ROS1. We have found that three putative helix-intercalating residues (Q607, R903 and M905) are required for processing of 5-meC:G pairs, but dispensable for excision of mismatched 5-meC. Mutant proteins Q607A, R903A and M905G retain the capacity to process an abasic site opposite G, thus suggesting that all three residues play a critical role in early steps of the base extrusion process and likely contribute to destabilization of 5-meC:G pairs. While R903 and M905 are not essential for DNA binding, mutation of Q607 abrogates stable binding to both methylated and nonmethylated DNA. However, the mutant protein Q607A can form stable complexes with DNA substrates containing blocked ends, which suggests that Q607 intercalates into the helix and inhibits sliding. Altogether, our results suggest that ROS1 uses three predicted helix-invading residues to actively interrogate DNA in search for 5-meC.
HnRNP A1 controls a splicing regulatory circuit promoting mesenchymal-to-epithelial transition [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Epithelial-to-mesenchymal transition (EMT) is an embryonic program used by cancer cells to acquire invasive capabilities becoming metastatic. Ron, a constitutively active isoform of the Ron tyrosine kinase receptor, arises from skipping of Ron exon 11 and provided the first example of an alternative splicing variant causatively linked to the activation of tumor EMT. Splicing of exon 11 is controlled by two adjacent regulatory elements, a silencer and an enhancer of splicing located in exon 12. The alternative splicing factor and oncoprotein SRSF1 directly binds to the enhancer, induces the production of Ron and activates EMT leading to cell locomotion. Interestingly, we now find an important role for hnRNP A1 in controlling the activity of the Ron silencer. HnRNP A1 is able to antagonize the binding of SRSF1 and prevent exon skipping. Notably, hnRNP A1, by inhibiting the production of Ron, activates the reversal program, namely the mesenchymal-to-epithelial transition, which instead occurs at the final metastasis sites. Also, hnRNP A1 affects Ron splicing by regulating the expression level of hnRNP A2/B1, which similarly to SRSF1 can promote Ron production. These results shed light on how splicing regulation contributes to the tumor progression and provide potential targets to develop anticancer therapies.
A competitive regulatory mechanism discriminates between juxtaposed splice sites and pri-miRNA structures [Nucleic Acids Research - recent issues] 2013-10-07 01:00
We have explored the functional relationships between spliceosome and Microprocessor complex activities in a novel class of microRNAs (miRNAs), named Splice site Overlapping (SO) miRNAs, whose pri-miRNA hairpins overlap splice sites. We focused on the evolutionarily conserved SO miR-34b, and we identified two indispensable elements for recognition of its 3' splice site: a branch point located in the hairpin and a downstream purine-rich exonic splicing enhancer. In minigene systems, splicing inhibition owing to exonic splicing enhancer deletion or AG 3'ss mutation increases miR-34b levels. Moreover, small interfering-mediated silencing of Drosha and/or DGCR8 improves splicing efficiency and abolishes miR-34b production. Thus, the processing of this 3' SO miRNA is regulated in an antagonistic manner by the Microprocessor and the spliceosome owing to competition between these two machineries for the nascent transcript. We propose that this novel mechanism is commonly used to regulate the relative amount of SO miRNA and messenger RNA produced from primary transcripts.
miRISC recruits decapping factors to miRNA targets to enhance their degradation [Nucleic Acids Research - recent issues] 2013-10-07 01:00
MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.
HCV IRES interacts with the 18S rRNA to activate the 40S ribosome for subsequent steps of translation initiation [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Previous analyses of complexes of 40S ribosomal subunits with the hepatitis C virus (HCV) internal ribosome entry site (IRES) have revealed contacts made by the IRES with ribosomal proteins. Here, using chemical probing, we show that the HCV IRES also contacts the backbone and bases of the CCC triplet in the 18S ribosomal RNA (rRNA) expansion segment 7. These contacts presumably provide interplay between IRES domain II and the AUG codon close to ribosomal protein S5, which causes a rearrangement of 18S rRNA structure in the vicinity of the universally conserved nucleotide G1639. As a result, G1639 becomes exposed and the corresponding site of the 40S subunit implicated in transfer RNA discrimination can select . These data are the first demonstration at nucleotide resolution of direct IRES–rRNA interactions and how they induce conformational transition in the 40S subunit allowing the HCV IRES to function without AUG recognition initiation factors.
Conservation of RNA chaperone activity of the human La-related proteins 4, 6 and 7 [Nucleic Acids Research - recent issues] 2013-10-07 01:00
The La module is a conserved tandem arrangement of a La motif and RNA recognition motif whose function has been best characterized in genuine La proteins. The best-characterized substrates of La proteins are pre-tRNAs, and previous work using tRNA mediated suppression in Schizosaccharomyces pombe has demonstrated that yeast and human La enhance the maturation of these using two distinguishable activities: UUU-3'OH-dependent trailer binding/protection and a UUU-3'OH independent activity related to RNA chaperone function. The La module has also been identified in several conserved families of La-related proteins (LARPs) that engage other RNAs, but their mode of RNA binding and function(s) are not well understood. We demonstrate that the La modules of the human LARPs 4, 6 and 7 are also active in tRNA-mediated suppression, even in the absence of stable UUU-3'OH trailer protection. Rather, the capacity of these to enhance pre-tRNA maturation is associated with RNA chaperone function, which we demonstrate to be a conserved activity for each hLARP in vitro. Our work reveals insight into the mechanisms by which La module containing proteins discriminate RNA targets and demonstrates that RNA chaperone activity is a conserved function across representative members of the La motif-containing superfamily.
Synthetic tolerance: three noncoding small RNAs, DsrA, ArcZ and RprA, acting supra-additively against acid stress [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Synthetic acid tolerance, especially during active cell growth, is a desirable phenotype for many biotechnological applications. Natively, acid resistance in Escherichia coli is largely a stationary-phase phenotype attributable to mechanisms mostly under the control of the stationary-phase sigma factor RpoS. We show that simultaneous overexpression of noncoding small RNAs (sRNAs), DsrA, RprA and ArcZ, which are translational RpoS activators, increased acid tolerance (based on a low-pH survival assay) supra-additively up to 8500-fold during active cell growth, and provided protection against carboxylic acid and oxidative stress. Overexpression of rpoS without its regulatory 5'-UTR resulted in inferior acid tolerance. The supra-additive effect of overexpressing the three sRNAs results from the impact their expression has on RpoS-protein levels, and the beneficial perturbation of the interconnected RpoS and H-NS networks, thus leading to superior tolerance during active growth. Unlike the overexpression of proteins, overexpression of sRNAs imposes hardly any metabolic burden on cells, and constitutes a more effective strain engineering strategy.
Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.
Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions [Nucleic Acids Research - recent issues] 2013-10-07 01:00
A p53 hot-spot mutation found frequently in human cancer is the replacement of R273 by histidine or cysteine residues resulting in p53 loss of function as a tumor suppressor. These mutants can be reactivated by the incorporation of second-site suppressor mutations. Here, we present high-resolution crystal structures of the p53 core domains of the cancer-related proteins, the rescued proteins and their complexes with DNA. The structures show that inactivation of p53 results from the incapacity of the mutated residues to form stabilizing interactions with the DNA backbone, and that reactivation is achieved through alternative interactions formed by the suppressor mutations. Detailed structural and computational analysis demonstrates that the rescued p53 complexes are not fully restored in terms of DNA structure and its interface with p53. Contrary to our previously studied wild-type (wt) p53-DNA complexes showing non-canonical Hoogsteen A/T base pairs of the DNA helix that lead to local minor-groove narrowing and enhanced electrostatic interactions with p53, the current structures display Watson–Crick base pairs associated with direct or water-mediated hydrogen bonds with p53 at the minor groove. These findings highlight the pivotal role played by R273 residues in supporting the unique geometry of the DNA target and its sequence-specific complex with p53.
Architecturally diverse proteins converge on an analogous mechanism to inactivate Uracil-DNA glycosylase [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Uracil-DNA glycosylase (UDG) compromises the replication strategies of diverse viruses from unrelated lineages. Virally encoded proteins therefore exist to limit, inhibit or target UDG activity for proteolysis. Viral proteins targeting UDG, such as the bacteriophage proteins ugi, and p56, and the HIV-1 protein Vpr, share no sequence similarity, and are not structurally homologous. Such diversity has hindered identification of known or expected UDG-inhibitory activities in other genomes. The structural basis for UDG inhibition by ugi is well characterized; yet, paradoxically, the structure of the unbound p56 protein is enigmatically unrevealing of its mechanism. To resolve this conundrum, we determined the structure of a p56 dimer bound to UDG. A helix from one of the subunits of p56 occupies the UDG DNA-binding cleft, whereas the dimer interface forms a hydrophobic box to trap a mechanistically important UDG residue. Surprisingly, these p56 inhibitory elements are unexpectedly analogous to features used by ugi despite profound architectural disparity. Contacts from B-DNA to UDG are mimicked by residues of the p56 helix, echoing the role of ugi’s inhibitory beta strand. Using mutagenesis, we propose that DNA mimicry by p56 is a targeting and specificity mechanism supporting tight inhibition via hydrophobic sequestration.
Solution structure of human P1*P2 heterodimer provides insights into the role of eukaryotic stalk in recruiting the ribosome-inactivating protein trichosanthin to the ribosome [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Lateral ribosomal stalk is responsible for binding and recruiting translation factors during protein synthesis. The eukaryotic stalk consists of one P0 protein with two copies of P1•P2 heterodimers to form a P0(P1•P2)2 pentameric P-complex. Here, we have solved the structure of full-length P1•P2 by nuclear magnetic resonance spectroscopy. P1 and P2 dimerize via their helical N-terminal domains, whereas the C-terminal tails of P1•P2 are unstructured and can extend up to ~125 Å away from the dimerization domains. 15N relaxation study reveals that the C-terminal tails are flexible, having a much faster internal mobility than the N-terminal domains. Replacement of prokaryotic L10(L7/L12)4/L11 by eukaryotic P0(P1•P2)2/eL12 rendered Escherichia coli ribosome, which is insensitive to trichosanthin (TCS), susceptible to depurination by TCS and the C-terminal tail was found to be responsible for this depurination. Truncation and insertion studies showed that depurination of hybrid ribosome is dependent on the length of the proline-alanine rich hinge region within the C-terminal tail. All together, we propose a model that recruitment of TCS to the sarcin-ricin loop required the flexible C-terminal tail, and the proline-alanine rich hinge region lengthens this C-terminal tail, allowing the tail to sweep around the ribosome to recruit TCS.
RNA duplexes with abasic substitutions are potent and allele-selective inhibitors of huntingtin and ataxin-3 expression [Nucleic Acids Research - recent issues] 2013-10-07 01:00
Abasic substitutions within DNA or RNA are tools for evaluating the impact of absent nucleobases. Because of the importance of abasic sites in genetic damage, most research has involved DNA. Little information is available on the impact of abasic substitutions within RNA or on RNA interference (RNAi). Here, we examine the effect of abasic substitutions on RNAi and allele-selective gene silencing. Huntington's disease (HD) and Machado Joseph Disease (MJD) are severe neurological disorders that currently have no cure. HD and MJD are caused by an expansion of CAG repeats within one mRNA allele encoding huntingtin (HTT) and ataxin-3 (ATX-3) proteins. Agents that silence mutant HTT or ATX-3 expression would remove the cause of HD or MJD and provide an option for therapeutic development. We describe flexible syntheses for abasic substitutions and show that abasic RNA duplexes allele-selectively inhibit both mutant HTT and mutant ATX-3. Inhibition involves the RNAi protein argonaute 2, even though the abasic substitution disrupts the catalytic cleavage of RNA target by argonaute 2. Several different abasic duplexes achieve potent and selective inhibition, providing a broad platform for subsequent development. These findings introduce abasic substitutions as a tool for tailoring RNA duplexes for gene silencing.
Nucleic Acids Research: VOLUME 41 ISSUE 17 2013 [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Nucleic Acids Research - Editorial Board [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Subscriptions [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Graph-based modeling of tandem repeats improves global multiple sequence alignment [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Tandem repeats (TRs) are often present in proteins with crucial functions, responsible for resistance, pathogenicity and associated with infectious or neurodegenerative diseases. This motivates numerous studies of TRs and their evolution, requiring accurate multiple sequence alignment. TRs may be lost or inserted at any position of a TR region by replication slippage or recombination, but current methods assume fixed unit boundaries, and yet are of high complexity. We present a new global graph-based alignment method that does not restrict TR unit indels by unit boundaries. TR indels are modeled separately and penalized using the phylogeny-aware alignment algorithm. This ensures enhanced accuracy of reconstructed alignments, disentangling TRs and measuring indel events and rates in a biologically meaningful way. Our method detects not only duplication events but also all changes in TR regions owing to recombination, strand slippage and other events inserting or deleting TR units. We evaluate our method by simulation incorporating TR evolution, by either sampling TRs from a profile hidden Markov model or by mimicking strand slippage with duplications. The new method is illustrated on a family of type III effectors, a pathogenicity determinant in agriculturally important bacteria Ralstonia solanacearum. We show that TR indel rate variation contributes to the diversification of this protein family.
An efficient strategy for TALEN-mediated genome engineering in Drosophila [Nucleic Acids Research - recent issues] 2013-09-25 08:09
In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis-regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila. We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F1 generation.
A genome-wide screen for identifying all regulators of a target gene [Nucleic Acids Research - recent issues] 2013-09-25 08:09
We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP–cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.
TrAp: a tree approach for fingerprinting subclonal tumor composition [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Revealing the clonal composition of a single tumor is essential for identifying cell subpopulations with metastatic potential in primary tumors or with resistance to therapies in metastatic tumors. Sequencing technologies provide only an overview of the aggregate of numerous cells. Computational approaches to de-mix a collective signal composed of the aberrations of a mixed cell population of a tumor sample into its individual components are not available. We propose an evolutionary framework for deconvolving data from a single genome-wide experiment to infer the composition, abundance and evolutionary paths of the underlying cell subpopulations of a tumor. We have developed an algorithm (TrAp) for solving this mixture problem. In silico analyses show that TrAp correctly deconvolves mixed subpopulations when the number of subpopulations and the measurement errors are moderate. We demonstrate the applicability of the method using tumor karyotypes and somatic hypermutation data sets. We applied TrAp to Exome-Seq experiment of a renal cell carcinoma tumor sample and compared the mutational profile of the inferred subpopulations to the mutational profiles of single cells of the same tumor. Finally, we deconvolve sequencing data from eight acute myeloid leukemia patients and three distinct metastases of one melanoma patient to exhibit the evolutionary relationships of their subpopulations.
Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts [Nucleic Acids Research - recent issues] 2013-09-25 08:09
It is a challenge to classify protein-coding or non-coding transcripts, especially those re-constructed from high-throughput sequencing data of poorly annotated species. This study developed and evaluated a powerful signature tool, Coding-Non-Coding Index (CNCI), by profiling adjoining nucleotide triplets to effectively distinguish protein-coding and non-coding sequences independent of known annotations. CNCI is effective for classifying incomplete transcripts and sense–antisense pairs. The implementation of CNCI offered highly accurate classification of transcripts assembled from whole-transcriptome sequencing data in a cross-species manner, that demonstrated gene evolutionary divergence between vertebrates, and invertebrates, or between plants, and provided a long non-coding RNA catalog of orangutan. CNCI software is available at http://www.bioinfo.org/software/cnci.
A new strategy for gene targeting and functional proteomics using the DT40 cell line [Nucleic Acids Research - recent issues] 2013-09-25 08:09
DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry–based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination–based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.
A multidimensional platform for the purification of non-coding RNA species [Nucleic Acids Research - recent issues] 2013-09-25 08:09
A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.
Detecting Alu insertions from high-throughput sequencing data [Nucleic Acids Research - recent issues] 2013-09-25 08:09
High-throughput sequencing technologies have allowed for the cataloguing of variation in personal human genomes. In this manuscript, we present alu-detect, a tool that combines read-pair and split-read information to detect novel Alus and their precise breakpoints directly from either whole-genome or whole-exome sequencing data while also identifying insertions directly in the vicinity of existing Alus. To set the parameters of our method, we use simulation of a faux reference, which allows us to compute the precision and recall of various parameter settings using real sequencing data. Applying our method to 100 bp paired Illumina data from seven individuals, including two trios, we detected on average 1519 novel Alus per sample. Based on the faux-reference simulation, we estimate that our method has 97% precision and 85% recall. We identify 808 novel Alus not previously described in other studies. We also demonstrate the use of alu-detect to study the local sequence and global location preferences for novel Alu insertions.
VEGF-A mRNA processing, stability and translation: a paradigm for intricate regulation of gene expression at the post-transcriptional level [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Vascular Endothelial Growth Factor A (VEGF-A) is a potent secreted mitogen crucial for physiological and pathological angiogenesis. Post-transcriptional regulation of VEGF-A occurs at multiple levels. Firstly, alternative splicing gives rise to different transcript variants encoding diverse isoforms that exhibit distinct biological properties with regard to receptor binding and extra-cellular localization. Secondly, VEGF-A mRNA stability is regulated by effectors such as hypoxia or growth factors through the binding of stabilizing and destabilizing proteins at AU-rich elements located in the 3'-untranslated region. Thirdly, translation of VEGF-A mRNA is a controlled process involving alternative initiation codons, internal ribosome entry sites (IRESs), an upstream open reading frame (uORF), miRNA targeting and a riboswitch in the 3' untranslated region. These different levels of regulation cooperate for the crucial fine-tuning of the expression of VEGF-A variants. This review will be focused on our current knowledge of the complex post-transcriptional regulatory switches that modulate the cellular VEGF-A level, a paradigmatic model of post-transcriptional regulation.
Dissecting cancer heterogeneity with a probabilistic genotype-phenotype model [Nucleic Acids Research - recent issues] 2013-09-25 08:09
One of the obstacles hindering a better understanding of cancer is its heterogeneity. However, computational approaches to model cancer heterogeneity have lagged behind. To bridge this gap, we have developed a new probabilistic approach that models individual cancer cases as mixtures of subtypes. Our approach can be seen as a meta-model that summarizes the results of a large number of alternative models. It does not assume predefined subtypes nor does it assume that such subtypes have to be sharply defined. Instead given a measure of phenotypic similarity between patients and a list of potential explanatory features, such as mutations, copy number variation, microRNA levels, etc., it explains phenotypic similarities with the help of these features. We applied our approach to Glioblastoma Multiforme (GBM). The resulting model Prob_GBM, not only correctly inferred known relationships but also identified new properties underlining phenotypic similarities. The proposed probabilistic framework can be applied to model relations between similarity of gene expression and a broad spectrum of potential genetic causes.
Translational sensitivity of the Escherichia coli genome to fluctuating tRNA availability [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The synthesis of protein from messenger RNA during translation is a highly dynamic process that plays a key role in controlling the efficiency and fidelity of genome-wide protein expression. The availability of aminoacylated transfer RNA (tRNA) is a major factor influencing the speed of ribosomal movement, which depending on codon choices, varies considerably along a transcript. Furthermore, it has been shown experimentally that tRNA availability can vary significantly under different growth and stress conditions, offering the cell a way to adapt translational dynamics across the genome. Existing models of translation have neglected fluctuations of tRNA pools, instead assuming fixed tRNA availabilities over time. This has lead to an incomplete understanding of this process. Here, we show for the entire Escherichia coli genome how and to what extent translational speed profiles, which capture local aspects of translational elongation, respond to measured shifts in tRNA availability. We find that translational profiles across the genome are affected to differing degrees, with genes that are essential or related to fundamental processes such as translation, being more robust than those linked to regulation. Furthermore, we reveal how fluctuating tRNA availability influences profiles of specific sequences known to play a significant role in translational control of gene expression.
CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap.
Mineralocorticoid receptor interaction with SP1 generates a new response element for pathophysiologically relevant gene expression [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1–MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.
Estrogen represses gene expression through reconfiguring chromatin structures [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Estrogen regulates over a thousand genes, with an equal number of them being induced or repressed. The distinct mechanisms underlying these dual transcriptional effects remain largely unknown. We derived comprehensive views of the transcription machineries assembled at estrogen-responsive genes through integrating multiple types of genomic data. In the absence of estrogen, the majority of genes formed higher-order chromatin structures, including DNA loops tethered to protein complexes involving RNA polymerase II (Pol II), estrogen receptor alpha (ERα) and ERα-pioneer factors. Genes to be ‘repressed’ by estrogen showed active transcription at promoters and throughout the gene bodies; genes to be ‘induced’ exhibited active transcription initiation at promoters, but with transcription paused in gene bodies. In the presence of estrogen, the majority of estrogen-induced genes retained the original higher-order chromatin structures, whereas most estrogen-repressed genes underwent a chromatin reconfiguration. For estrogen-induced genes, estrogen enhances transcription elongation, potentially through recruitment of co-activators or release of co-repressors with unique roles in elongation. For estrogen-repressed genes, estrogen treatment leads to chromatin structure reconfiguration, thereby disrupting the originally transcription-efficient chromatin structures. Our in silico studies have shown that estrogen regulates gene expression, at least in part, through modifying previously assembled higher-order complexes, rather than by facilitating de novo assembly of machineries.
Genomic targets, and histone acetylation and gene expression profiling of neural HDAC inhibition [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Histone deacetylase inhibitors (HDACis) have been shown to potentiate hippocampal-dependent memory and synaptic plasticity and to ameliorate cognitive deficits and degeneration in animal models for different neuropsychiatric conditions. However, the impact of these drugs on hippocampal histone acetylation and gene expression profiles at the genomic level, and the molecular mechanisms that underlie their specificity and beneficial effects in neural tissue, remains obscure. Here, we mapped four relevant histone marks (H3K4me3, AcH3K9,14, AcH4K12 and pan-AcH2B) in hippocampal chromatin and investigated at the whole-genome level the impact of HDAC inhibition on acetylation profiles and basal and activity-driven gene expression. HDAC inhibition caused a dramatic histone hyperacetylation that was largely restricted to active loci pre-marked with H3K4me3 and AcH3K9,14. In addition, the comparison of Chromatin immunoprecipitation sequencing and gene expression profiles indicated that Trichostatin A-induced histone hyperacetylation, like histone hypoacetylation induced by histone acetyltransferase deficiency, had a modest impact on hippocampal gene expression and did not affect the transient transcriptional response to novelty exposure. However, HDAC inhibition caused the rapid induction of a homeostatic gene program related to chromatin deacetylation. These results illuminate both the relationship between hippocampal gene expression and histone acetylation and the mechanism of action of these important neuropsychiatric drugs.
PP1{alpha}, PP1{beta} and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3 [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo, whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo. These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.
Estrogen receptor {alpha} can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.
Deciphering the modulation of gene expression by type I and II interferons combining 4sU-tagging, translational arrest and in silico promoter analysis [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Interferons (IFN) play a pivotal role in innate immunity, orchestrating a cell-intrinsic anti-pathogenic state and stimulating adaptive immune responses. The complex interplay between the primary response to IFNs and its modulation by positive and negative feedback loops is incompletely understood. Here, we implement the combination of high-resolution gene-expression profiling of nascent RNA with translational inhibition of secondary feedback by cycloheximide. Unexpectedly, this approach revealed a prominent role of negative feedback mechanisms during the immediate (≤60 min) IFNα response. In contrast, a more complex picture involving both negative and positive feedback loops was observed on IFN treatment. IFN-induced repression of genes associated with regulation of gene expression, cellular development, apoptosis and cell growth resulted from cycloheximide-resistant primary IFN signalling. In silico promoter analysis revealed significant overrepresentation of SP1/SP3-binding sites and/or GC-rich stretches. Although signal transducer and activator of transcription 1 (STAT1)-binding sites were not overrepresented, repression was lost in absence of STAT1. Interestingly, basal expression of the majority of these IFN-repressed genes was dependent on STAT1 in IFN-naïve fibroblasts. Finally, IFN-mediated repression was also found to be evident in primary murine macrophages. IFN-repressed genes include negative regulators of innate and stress response, and their decrease may thus aid the establishment of a signalling perceptive milieu.
The Igf2/H19 muscle enhancer is an active transcriptional complex [Nucleic Acids Research - recent issues] 2013-09-25 08:09
In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1. Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.
Global 'bootprinting' reveals the elastic architecture of the yeast TFIIIB-TFIIIC transcription complex in vivo [Nucleic Acids Research - recent issues] 2013-09-25 08:09
TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB–TFIIIC complexes in Saccharomyces cerevisiae, obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed ‘bootprints’. Global analysis reveals that the TFIIIB–TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site (ETC10). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.
An intronic structure enabled by a long-distance interaction serves as a novel target for splicing correction in spinal muscular atrophy [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Here, we report a long-distance interaction (LDI) as a critical regulator of alternative splicing of Survival Motor Neuron 2 (SMN2) exon 7, skipping of which is linked to spinal muscular atrophy (SMA), a leading genetic disease of children and infants. We show that this LDI is linked to a unique intra-intronic structure that we term internal stem through LDI-1 (ISTL1). We used site-specific mutations and Selective 2'-Hydroxyl Acylation analyzed by Primer Extension to confirm the formation and functional significance of ISTL1. We demonstrate that the inhibitory effect of ISTL1 is independent of hnRNP A1/A2B1 and PTB1 previously implicated in SMN2 exon 7 splicing. We show that an antisense oligonucleotide-mediated sequestration of the 3' strand of ISTL1 fully corrects SMN2 exon 7 splicing and restores high levels of SMN and Gemin2, a SMN-interacting protein, in SMA patient cells. Our results also reveal that the 3' strand of ISTL1 and upstream sequences constitute an inhibitory region that we term intronic splicing silencer N2 (ISS-N2). This is the first report to demonstrate a critical role of a structure-associated LDI in splicing regulation of an essential gene linked to a genetic disease. Our findings expand the repertoire of potential targets for an antisense oligonucleotide-mediated therapy of SMA.
Using stable MutS dimers and tetramers to quantitatively analyze DNA mismatch recognition and sliding clamp formation [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.
Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1 [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Strand displacement by a DNA polymerase serves a key role in Okazaki fragment maturation, which involves displacement of the RNA primer of the preexisting Okazaki fragment into a flap structure, and subsequent flap removal and fragment ligation. We investigated the role of Sulfolobus chromatin proteins Sso7d and Cren7 in strand displacement by DNA polymerase B1 (PolB1) from the hyperthermophilic archaeon Sulfolobus solfataricus. PolB1 showed a robust strand displacement activity and was capable of synthesizing thousands of nucleotides on a DNA-primed 72-nt single-stranded circular DNA template. This activity was inhibited by both Sso7d and Cren7, which limited the flap length to 3–4 nt at saturating concentrations. However, neither protein inhibited RNA displacement on an RNA-primed single-stranded DNA minicircle by PolB1. Strand displacement remained sensitive to modulation by the chromatin proteins when PolB1 was in association with proliferating cell nuclear antigen. Inhibition of DNA instead of RNA strand displacement by the chromatin proteins is consistent with the finding that double-stranded DNA was more efficiently bound and stabilized than an RNA:DNA duplex by these proteins. Our results suggest that Sulfolobus chromatin proteins modulate strand displacement by PolB1, permitting efficient removal of the RNA primer while inhibiting excessive displacement of the newly synthesized DNA strand during Okazaki fragment maturation.
Involvement of Schizosaccharomyces pombe rrp1+ and rrp2+ in the Srs2- and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe. Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.
Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.
Widespread purifying selection on RNA structure in mammals [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies >4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5–22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1. This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes.
Widespread evidence of cooperative DNA binding by transcription factors in Drosophila development [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that >65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.
Rationally designed coiled-coil DNA looping peptides control DNA topology [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein–DNA nanostructures.
Non-FG mediated transport of the large pre-ribosomal subunit through the nuclear pore complex by the mRNA export factor Gle2 [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Multiple export receptors passage bound pre-ribosomes through nuclear pore complexes (NPCs) by transiently interacting with the Phe-Gly (FG) meshwork of their transport channels. Here, we reveal how the non-FG interacting yeast mRNA export factor Gly-Leu-FG lethal 2 (Gle2) functions in the export of the large pre-ribosomal subunit (pre-60S). Structure-guided studies uncovered conserved platforms used by Gle2 to export pre-60S: an uncharacterized basic patch required to bind pre-60S, and a second surface that makes non-FG contacts with the nucleoporin Nup116. A basic patch mutant of Gle2 is able to function in mRNA export, but not pre-60S export. Thus, Gle2 provides a distinct interaction platform to transport pre-60S to the cytoplasm. Notably, Gle2’s interaction platforms become crucial for pre-60S export when FG-interacting receptors are either not recruited to pre-60S or are impaired. We propose that large complex cargos rely on non-FG as well as FG-interactions for their efficient translocation through the nuclear pore complex channel.
Time-dependent bending rigidity and helical twist of DNA by rearrangement of bound HU protein [Nucleic Acids Research - recent issues] 2013-09-25 08:09
HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ~2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.
Characterization of PA-N terminal domain of Influenza A polymerase reveals sequence specific RNA cleavage [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Influenza virus uses a unique cap-snatching mechanism characterized by hijacking and cleavage of host capped pre-mRNAs, resulting in short capped RNAs, which are used as primers for viral mRNA synthesis. The PA subunit of influenza polymerase carries the endonuclease activity that catalyzes the host mRNA cleavage reaction. Here, we show that PA is a sequence selective endonuclease with distinct preference to cleave at the 3' end of a guanine (G) base in RNA. The G specificity is exhibited by the native influenza polymerase complex associated with viral ribonucleoprotein particles and is conferred by an intrinsic G specificity of the isolated PA endonuclease domain PA-Nter. In addition, RNA cleavage site choice by the full polymerase is also guided by cap binding to the PB2 subunit, from which RNA cleavage preferentially occurs at the 12th nt downstream of the cap. However, if a G residue is present in the region of 10–13 nucleotides from the cap, cleavage preferentially occurs at G. This is the first biochemical evidence of influenza polymerase PA showing intrinsic sequence selective endonuclease activity.
Magnetic isotope and magnetic field effects on the DNA synthesis [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Magnetic isotope and magnetic field effects on the rate of DNA synthesis catalysed by polymerases β with isotopic ions 24Mg2+, 25Mg2+ and 26Mg2+ in the catalytic sites were detected. No difference in enzymatic activity was found between polymerases β carrying 24Mg2+ and 26Mg2+ ions with spinless, non-magnetic nuclei 24Mg and 26Mg. However, 25Mg2+ ions with magnetic nucleus 25Mg were shown to suppress enzymatic activity by two to three times with respect to the enzymatic activity of polymerases β with 24Mg2+ and 26Mg2+ ions. Such an isotopic dependence directly indicates that in the DNA synthesis magnetic mass-independent isotope effect functions. Similar effect is exhibited by polymerases β with Zn2+ ions carrying magnetic 67Zn and non-magnetic 64Zn nuclei, respectively. A new, ion–radical mechanism of the DNA synthesis is suggested to explain these effects. Magnetic field dependence of the magnesium-catalysed DNA synthesis is in a perfect agreement with the proposed ion–radical mechanism. It is pointed out that the magnetic isotope and magnetic field effects may be used for medicinal purposes (trans-cranial magnetic treatment of cognitive deceases, cell proliferation, control of the cancer cells, etc).
Dyskerin depletion increases VEGF mRNA internal ribosome entry site-mediated translation [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Dyskerin is a nucleolar protein encoded by the DKC1 gene that (i) stabilizes the RNA component of the telomerase complex, and (ii) drives the site-specific pseudouridilation of rRNA. It is known that the partial lack of dyskerin function causes a defect in the translation of a subgroup of mRNAs containing internal ribosome entry site (IRES) elements such as those encoding for the tumor suppressors p27 and p53. In this study, we aimed to analyze what is the effect of the lack of dyskerin on the IRES-mediated translation of mRNAs encoding for vascular endothelial growth factor (VEGF). We transiently reduced dyskerin expression and measured the levels of the IRES-mediated translation of the mRNA encoding for VEGF in vitro in transformed and primary cells. We demonstrated a significant increase in the VEGF IRES-mediated translation after dyskerin knock-down. This translational modulation induces an increase in VEGF production in the absence of a significant upregulation in VEGF mRNA levels. The analysis of a list of viral and cellular IRESs indicated that dyskerin depletion can differentially affect IRES-mediated translation. These results indicate for the first time that dyskerin inhibition can upregulate the IRES translation initiation of specific mRNAs.
DHX34 and NBAS form part of an autoregulatory NMD circuit that regulates endogenous RNA targets in human cells, zebrafish and Caenorhabditis elegans [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species. Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.
Modification of the RpoS network with a synthetic small RNA [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Translation of the sigma factor RpoS is activated by DsrA, RprA and ArcA, three small non-coding sRNAs (sRNA) that expose the ribosome-binding site (RBS) by opening up an inhibitory loop. In the RpoS network, no sRNAs have been found to pair with the RBS, a most common sRNA target site in bacteria. Here, we generate Ribo-0, an artificial sRNA, which represses rpoS translation by pairing with the RBS. Ribo-0 bypasses the RNA chaperon Hfq but requires the RBS to be loosely blocked. Ribo-0 interacts with DsrA and reshapes the RpoS network. Specifically, in the intact RpoS network, DsrA activates rpoS translation by freeing up the RBS. In the modified RpoS network where Ribo-0 is introduced, the DsrA-caused RBS exposure facilitates Ribo-0 binding, thereby strengthening Ribo-0 inhibition. In other words, Ribo-0 changes DsrA from an activator to an accomplice for repressing rpoS translation. This work presents an artificial mechanism of rpoS regulation, reveals mutual effects of native and synthetic players and demonstrates genetic context-dependency of their functions.
Attachment site recognition and regulation of directionality by the serine integrases [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Serine integrases catalyze the integration of bacteriophage DNA into a host genome by site-specific recombination between ‘attachment sites’ in the phage (attP) and the host (attB). The reaction is highly directional; the reverse excision reaction between the product attL and attR sites does not occur in the absence of a phage-encoded factor, nor does recombination occur between other pairings of attachment sites. A mechanistic understanding of how these enzymes achieve site-selectivity and directionality has been limited by a lack of structural models. Here, we report the structure of the C-terminal domains of a serine integrase bound to an attP DNA half-site. The structure leads directly to models for understanding how the integrase-bound attP and attB sites differ, why these enzymes preferentially form attP x attB synaptic complexes to initiate recombination, and how attL x attR recombination is prevented. In these models, different domain organizations on attP vs. attB half-sites allow attachment-site specific interactions to form between integrase subunits via an unusual protruding coiled-coil motif. These interactions are used to preferentially synapse integrase-bound attP and attB and inhibit synapsis of integrase-bound attL and attR. The results provide a structural framework for understanding, testing and engineering serine integrase function.
The structure of Escherichia coli ExoIX--implications for DNA binding and catalysis in flap endonucleases [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.
Structure of p53 binding to the BAX response element reveals DNA unwinding and compression to accommodate base-pair insertion [Nucleic Acids Research - recent issues] 2013-09-25 08:09
The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.
Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions [Nucleic Acids Research - recent issues] 2013-09-25 08:09
Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5' cap structure is a crucial step that commits deadenylated mRNAs to 5'-to-3' degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking–mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.
Dystrophin rescue by trans-splicing: a strategy for DMD genotypes not eligible for exon skipping approaches [Nucleic Acids Research - recent issues] 2013-09-25 08:09
RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans-splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre–trans-splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans-splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv, illustrating that a given PTM can be suitable for a variety of mutations.
Visualizing genomic information across chromosomes with PhenoGram [BioData Mining - Latest Articles] 2013-10-15 20:00
Background: With the abundance of information and analysis results being collected for genetic loci, user-friendly and flexible data visualization approaches can inform and improve the analysis and dissemination of these data. A chromosomal ideogram is an idealized graphic representation of chromosomes. Ideograms can be combined with overlaid points, lines, and/or shapes, to provide summary information from studies of various kinds, such as genome-wide association studies or phenome-wide association studies, coupled with genomic location information. To facilitate visualizing varied data in multiple ways using ideograms, we have developed a flexible software tool called PhenoGram which exists as a web-based tool and also a command-line program. Results: With PhenoGram researchers can create chomosomal ideograms annotated with lines in color at specific base-pair locations, or colored base-pair to base-pair regions, with or without other annotation. PhenoGram allows for annotation of chromosomal locations and/or regions with shapes in different colors, gene identifiers, or other text. PhenoGram also allows for creation of plots showing expanded chromosomal locations, providing a way to show results for specific chromosomal regions in greater detail. We have now used PhenoGram to produce a variety of different plots, and provide these as examples herein. These plots include visualization of the genomic coverage of SNPs from a genotyping array, highlighting the chromosomal coverage of imputed SNPs, copy-number variation region coverage, as well as plots similar to the NHGRI GWA Catalog of genome-wide association results. Conclusions: PhenoGram is a versatile, user-friendly software tool fostering the exploration and sharing of genomic information. Through visualization of data, researchers can both explore and share complex results, facilitating a greater understanding of these data.
Using random walks to identify cancer-associated modules in expression data [BioData Mining - Latest Articles] 2013-10-14 20:00
Background: The etiology of cancer involves a complex series of genetic and environmental conditions. To better represent and study the intricate genetics of cancer onset and progression, we construct a network of biological interactions to search for groups of genes that compose cancer-related modules. Three cancer expression datasets are investigated to prioritize genes and interactions associated with cancer outcomes. Using a graph-based approach to search for communities of phenotype-related genes in microarray data, we find modules of genes associated with cancer phenotypes in a weighted interaction network. Results: We implement Walktrap, a random-walk-based community detection algorithm, to identify biological modules predisposing to tumor growth in 22 hepatocellular carcinoma samples (GSE14520), adenoma development in 32 colorectal cancer samples (GSE8671), and prognosis in 198 breast cancer patients (GSE7390). For each study, we find the best scoring partitions under a maximum cluster size of 200 nodes. Significant modules highlight groups of genes that are functionally related to cancer and show promise as therapeutic targets; these include interactions among transcription factors (SPIB, RPS6KA2 and RPS6KA6), cell-cycle regulatory genes (BRSK1, WEE1 and CDC25C), modulators of the cell-cycle and proliferation (CBLC and IRS2) and genes that regulate and participate in the map-kinase pathway (MAPK9, DUSP1, DUSP9, RIPK2). To assess the performance of Walktrap to find genomic modules (Walktrap-GM), we evaluate our results against other tools recently developed to discover disease modules in biological networks. Compared with other highly cited module-finding tools, jActiveModules and Matisse, Walktrap-GM shows strong performance in the discovery of modules enriched with known cancer genes. Conclusions: These results demonstrate that the Walktrap-GM algorithm identifies modules significantly enriched with cancer genes, their joint effects and promising candidate genes. The approach performs well when evaluated against similar tools and smaller overall module size allows for more specific functional annotation and facilitates the interpretation of these modules.
LVQ-SMOTE ¿Learning Vector Quantization based Synthetic Minority Over¿sampling Technique for biomedical data [BioData Mining - Latest Articles] 2013-10-01 20:00
Background: Over-sampling methods based on Synthetic Minority Over-sampling Technique (SMOTE) have beenproposed for classification problems of imbalanced biomedical data. However, the existing oversamplingmethods achieve slightly better or sometimes worse result than the simplest SMOTE. In orderto improve the effectiveness of SMOTE, this paper presents a novel over-sampling method usingcodebooks obtained by the learning vector quantization. In general, even when an existing SMOTEapplied to a biomedical dataset, its empty feature space is still so huge that most classification algorithmswould not perform well on estimating borderlines between classes. To tackle this problem, ourover-sampling method generates synthetic samples which occupy more feature space than the otherSMOTE algorithms. Briefly saying, our over-sampling method enables to generate useful syntheticsamples by referring to actual samples taken from real-world datasets. Results: Experiments on eight real-world imbalanced datasets demonstrate that our proposed over-samplingmethod performs better than the simplest SMOTE on four of five standard classification algorithms.Moreover, it is seen that the performance of our method increases if the latest SMOTE called MWMOTEis used in our algorithm. Experiments on datasets for ß-turn types prediction show someimportant patterns that have not been seen in previous analyses. Conclusions: The proposed over-sampling method generates useful synthetic samples for the classification of imbalancedbiomedical data. Besides, the proposed over-sampling method is basically compatible withbasic classification algorithms and the existing over-sampling methods.
gff2sequence, a new user friendly tool for the generation of genomic sequences [BioData Mining - Latest Articles] 2013-09-10 20:00
Background: General Feature Format (GFF) files are used to store genome features such as genes, exons, introns, primary transcripts etc. Although many software packages (i.e. ab initio gene prediction programs) can annotate features by using such a standard, a small number of tools have been developed to extract the corresponding sequence information from the original genome. However the present tools do not execute either a quality control or a customizable filter of the annotated features is available.Findingsgff2sequence is a program that extracts nucleotide/protein sequences from a genomic multifasta by using the information provided by a general feature format file. While a graphical user interface makes this software very easy to use, a C++ algorithm allows high performance together with low hardware demand. The software also allows the extraction of the genic portions such as the untranslated and the coding sequences. Moreover a highly customizable quality control pipeline can be used to deal with anomalous splicing sites, incorrect open reading frames and not canonical characters within the retrieved sequences. Conclusions: gff2sequence is a user friendly program that allows the generation of highly customizable sequence datasets by processing a general feature format file. The presence of a wide range of quality filters makes this tool also suitable for refining the ab initio gene predictions.
The central role of biological data mining in connecting diverse disciplines [BioData Mining - Latest Articles] 2013-08-11 20:00
Scientific research has always suffered from the compartmentalization of different disciplines. This historic phenomenon is beginning to erode in specific broader disciplines such as medicine where new interdisciplinary areas such as bioinformatics have worked to bring together biologists with computer scientists and mathematicians. This is an encouraging trend but has yet to percolate to more disparate disciplines where there is tremendous potential for scientific advances. We had the pleasure and honor of participating in a 2013 Indonesian-American Kavli Frontiers of Science Symposium organized and sponsored jointly by the United States National Academy of Sciences and the Indonesian Academy of Sciences. This invitation-only scientific conference took place in Bali, Indonesia and was attended by young or early career scientists (Kavli Fellows) doing cutting-edge research in extremely diverse disciplines. The goals of the conference were two-fold. The first goal of was to expose researchers to very broad research topics to promote new research ideas within their own field or to expand their research into new fields. The second goal was to stimulate collaboration between U.S. and Indonesian researchers to foster a global exchange of ideas.
Unraveling genomic variation from next generation sequencing data [BioData Mining - Latest Articles] 2013-07-24 20:00
Elucidating the content of a DNA sequence is critical to deeper understand and decode the genetic information for any biological system. As next generation sequencing (NGS) techniques have become cheaper and more advanced in throughput over time, great innovations and breakthrough conclusions have been generated in various biological areas. Few of these areas, which get shaped by the new technological advances, involve evolution of species, microbial mapping, population genetics, genome-wide association studies (GWAs), comparative genomics, variant analysis, gene expression, gene regulation, epigenetics and personalized medicine. While NGS techniques stand as key players in modern biological research, the analysis and the interpretation of the vast amount of data that gets produced is a not an easy or a trivial task and still remains a great challenge in the field of bioinformatics. Therefore, efficient tools to cope with information overload, tackle the high complexity and provide meaningful visualizations to make the knowledge extraction easier are essential. In this article, we briefly refer to the sequencing methodologies and the available equipment to serve these analyses and we describe the data formats of the files which get produced by them. We conclude with a thorough review of tools developed to efficiently store, analyze and visualize such data with emphasis in structural variation analysis and comparative genomics. We finally comment on their functionality, strengths and weaknesses and we discuss how future applications could further develop in this field.
The disconnect between classical biostatistics and the biological data mining community [BioData Mining - Latest Articles] 2013-07-23 20:00
Statistics departments and journals still strongly emphasize a very narrow range of topics and methods and techniques, all driven by a tiny handful of results, many dating from the 1930s. Those methods may well have been good and amazing and quite appropriate for the available computing, known mathematical facts, and data of their day. Hence the common list of assumptions: normal distributions and very small parametric models and linearity and independent features. But the usual claims for these anchoring assumptions are accurate--when precisely true--but more often just irrelevant: data is rarely normal, model misspecification is always at work, features are highly entangled with functionally mysterious interactions, and multiple scientifically plausible models may all fit the data equally well. We describe here machine learning as an alternative.
MicroRNA-mediated regulation of target genes in several brain regions is correlated to both microRNA-targeting-specific promoter methylation and differential microRNA expression [BioData Mining - Latest Articles] 2013-05-30 20:00
Background: Public domain databases nowadays provide multiple layers of genome-wide data e.g., promoter methylation, mRNA expression, and miRNA expression and should enable integrative modeling of the mechanisms of regulation of gene expression. However, researches along this line were not frequently executed. Results: Here, the public domain dataset of mRNA expression, microRNA (miRNA) expression and promoter methylation patterns in four regions, the frontal cortex, temporal cortex, pons and cerebellum, of human brain were sourced from the National Center for Biotechnology Informations gene expression omnibus, and reanalyzed computationally. A large number of miRNA-mediated regulation of target genes and miRNA-targeting-specific promoter methylation were identified in the six pairwise comparisons among the four brain regions. The miRNA-mediated regulation of target genes was found to be highly correlated with one or both of miRNA-targeting-specific promoter methylation and differential miRNA expression. Genes enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways that were related to brain function and/or development were found among the target genes of miRNAs whose differential expression patterns were highly correlated with the miRNA-mediated regulation of their target genes. Conclusions: The combinatorial analysis of miRNA-mediated regulation of target genes, miRNA-targeting-specific promoter methylation and differential miRNA expression can help reveal the brain region-specific contributions of miRNAs to brain function and development.
The limits of p-values for biological data mining [BioData Mining - Latest Articles] 2013-05-10 20:00
This insightful Editorial discusses the applications and interpretations of p-values underlying analytical approaches in data mining and how they can be more complex than one might assume.
A robustness study of parametric and non-parametric tests in model-based multifactor dimensionality reduction for epistasis detection [BioData Mining - Latest Articles] 2013-04-24 20:00
Background: Applying a statistical method implies identifying underlying (model) assumptions and checking their validity in the particular context. One of these contexts is association modeling for epistasis detection. Here, depending on the technique used, violation of model assumptions may result in increased type I error, power loss, or biased parameter estimates. Remedial measures for violated underlying conditions or assumptions include data transformation or selecting a more relaxed modeling or testing strategy. Model-Based Multifactor Dimensionality Reduction (MB-MDR) for epistasis detection relies on association testing between a trait and a factor consisting of multilocus genotype information. For quantitative traits, the framework is essentially Analysis of Variance (ANOVA) that decomposes the variability in the trait amongst the different factors. In this study, we assess through simulations, the cumulative effect of deviations from normality and homoscedasticity on the overall performance of quantitative Model-Based Multifactor Dimensionality Reduction (MB-MDR) to detect 2-locus epistasis signals in the absence of main effects.MethodologyOur simulation study focuses on pure epistasis models with varying degrees of genetic influence on a quantitative trait. Conditional on a multilocus genotype, we consider quantitative trait distributions that are normal, chi-square or Student’s t with constant or non-constant phenotypic variances. All data are analyzed with MB-MDR using the built-in Student’s t-test for association, as well as a novel MB-MDR implementation based on Welch’s t-test. Traits are either left untransformed or are transformed into new traits via logarithmic, standardization or rank-based transformations, prior to MB-MDR modeling. Results: Our simulation results show that MB-MDR controls type I error and false positive rates irrespective of the association test considered. Empirically-based MB-MDR power estimates for MB-MDR with Welch’s t-tests are generally lower than those for MB-MDR with Student’s t-tests. Trait transformations involving ranks tend to lead to increased power compared to the other considered data transformations. Conclusions: When performing MB-MDR screening for gene-gene interactions with quantitative traits, we recommend to first rank-transform traits to normality and then to apply MB-MDR modeling with Student’s t-tests as internal tests for association.
Identification of properties important to protein aggregation using feature selection [BMC Bioinformatics - Latest Articles] 2013-10-27 20:00
Background: Protein aggregation is a significant problem in the biopharmaceutical industry (protein drug stability) and is associated medically with over 40 human diseases. Although a number of computational models have been developed for predicting aggregation propensity and identifying aggregation-prone regions in proteins, little systematic research has been done to determine physicochemical properties relevant to aggregation and their relative importance to this important process. Such studies may result in not only accurately predicting peptide aggregation propensities and identifying aggregation prone regions in proteins, but also aid in discovering additional underlying mechanisms governing this process. Results: We use two feature selection algorithms to identify 16 features, out of a total of 560 physicochemical properties, presumably important to protein aggregation. Two predictors (ProA-SVM and ProA-RF) using selected features are built for predicting peptide aggregation propensity and identifying aggregation prone regions in proteins. Both methods are compared favourably to other state-of-the-art algorithms in cross validation. The identified important properties are fairly consistent with previous studies and bring some new insights into protein and peptide aggregation. One interesting new finding is that aggregation prone peptide sequences have similar properties to signal peptide and signal anchor sequences. Conclusions: Both predictors are implemented in a freely available web application (http://www.abl.ku.edu/ProA/). We suggest that the quaternary structure of protein aggregates, especially soluble oligomers, may allow the formation of new molecular recognition signals that guide aggregate targeting to specific cellular sites.
Compact representation of k-mer de Bruijn graphs for genome read assembly [BMC Bioinformatics - Latest Articles] 2013-10-22 20:00
Background: Processing of reads from high throughput sequencing is often done in terms of edges in the de Bruijn graph representing all k-mers from the reads. The memory requirements for storing all k-mers in a lookup table can be demanding, even after removal of read errors, but can be alleviated by using a memory efficient data structure. Results: The FM-index, which is based on the Burrows¿Wheeler transform, provides an efficient data structure providing a searchable index of all substrings from a set of strings, and is used to compactly represent full genomes for use in mapping reads to a genome: the memory required to store this is in the same order of magnitude as the strings themselves. However, reads from high throughput sequences mostly have high coverage and so contain the same substrings multiple times from different reads. I here present a modification of the FM-index, which I call the kFM-index, for indexing the set of k-mers from the reads. For DNA sequences, this requires 5 bit of information for each vertex of the corresponding de Bruijn subgraph, i.e. for each different k-1-mer, plus some additional overhead, typically 0.5 to 1 bit per vertex, for storing the equivalent of the FM-index for walking the underlying de Bruijn graph and reproducing the actual k-mers efficiently. Conclusions: The kFM-index could replace more memory demanding data structures for storing the de Bruijn k-mer graph representation of sequence reads. A Java implementation with additional technical documentation is provided which demonstrates the applicability of the data structure (http://folk.uio.no/einarro/Projects/KFM-index/).
A scalable, knowledge-based analysis framework for genetic association studies [BMC Bioinformatics - Latest Articles] 2013-10-22 20:00
Background: Testing for marginal associations between numerous genetic variants and disease may miss complex relationships among variables (e.g., gene-gene interactions). Bayesian approaches can model multiple variables together and offer advantages over conventional model building strategies, including using existing biological evidence as modeling priors and acknowledging that many models may fit the data well. With many candidate variables, Bayesian approaches to variable selection rely on algorithms to approximate the posterior distribution of models, such as Markov-Chain Monte Carlo (MCMC). Unfortunately, MCMC is difficult to parallelize and requires many iterations to adequately sample the posterior. We introduce a scalable algorithm called PEAK that improves the efficiency of MCMC by dividing a large set of variables into related groups using a rooted graph that resembles a mountain peak. Our algorithm takes advantage of parallel computing and existing biological databases when available. Results: By using graphs to manage a model space with more than 500,000 candidate variables, we were able to improve MCMC efficiency and uncover the true simulated causal variables, including a gene-gene interaction. We applied PEAK to a case-control study of childhood asthma with 2,521 genetic variants. We used an informative graph for oxidative stress derived from Gene Ontology and identified several variants in ERBB4, OXR1, and BCL2 with strong evidence for associations with childhood asthma. Conclusions: We introduced an extremely flexible analysis framework capable of efficiently performing Bayesian variable selection on many candidate variables. The PEAK algorithm can be provided with an informative graph, which can be advantageous when considering gene-gene interactions, or a symmetric graph, which simply divides the model space into manageable regions. The PEAK framework is compatible with various model forms, allowing for the algorithm to be configured for different study designs and applications, such as pathway or rare-variant analyses, by simple modifications to the model likelihood and proposal functions.
Parametric sensitivity analysis for biochemical reaction networks based on pathwise information theory [BMC Bioinformatics - Latest Articles] 2013-10-21 20:00
Background: Stochastic modeling and simulation provide powerful predictive methods for the intrinsic understanding of fundamental mechanisms in complex biochemical networks. Typically, such mathematical models involve networks of coupled jump stochastic processes with a large number of parameters that need to be suitably calibrated against experimental data. In this direction, the parameter sensitivity analysis of reaction networks is an essential mathematical and computational tool, yielding information regarding the robustness and the identifiability of model parameters. However, existing sensitivity analysis approaches such as variants of the finite difference method can have an overwhelming computational cost in models with a high-dimensional parameter space. Results: We develop a sensitivity analysis methodology suitable for complex stochastic reaction networks with a large number of parameters. The proposed approach is based on Information Theory methods and relies on the quantification of information loss due to parameter perturbations between time-series distributions. For this reason, we need to work on path-space, i.e., the set consisting of all stochastic trajectories, hence the proposed approach is referred to as "pathwise". The pathwise sensitivity analysis method is realized by employing the rigorously-derived Relative Entropy Rate, which is directly computable from the propensity functions. A key aspect of the method is that an associated pathwise Fisher Information Matrix (FIM) is defined, which in turn constitutes a gradient-free approach to quantifying parameter sensitivities. The structure of the FIM turns out to be block-diagonal, revealing hidden parameter dependencies and sensitivities in reaction networks. Conclusions: As a gradient-free method, the proposed sensitivity analysis provides a significant advantage when dealing with complex stochastic systems with a large number of parameters. In addition, the knowledge of the structure of the FIM can allow to efficiently address questions on parameter identifiability, estimation and robustness. The proposed method is tested and validated on three biochemical systems, namely: (a) a protein production/degradation model where explicit solutions are available, permitting a careful assessment of the method, (b) the p53 reaction network where quasi-steady stochastic oscillations of the concentrations are observed, and for which continuum approximations (e.g. mean field, stochastic Langevin, etc.) break down due to persistent oscillations between high and low populations, and (c) an Epidermal Growth Factor Receptor model which is an example of a high-dimensional stochastic reaction network with more than 200 reactions and a corresponding number of parameters.
A method to identify differential expression profiles of time-course gene data with Fourier transformation [BMC Bioinformatics - Latest Articles] 2013-10-17 20:00
Background: Time course gene expression experiments are an increasingly popular method for exploring biological processes. Temporal gene expression profiles provide an important characterization of gene function, as biological systems are both developmental and dynamic. With such data it is possible to study gene expression changes over time and thereby to detect differential genes. Much of the early work on analyzing time series expression data relied on methods developed originally for static data and thus there is a need for improved methodology. Since time series expression is a temporal process, its unique features such as autocorrelation between successive points should be incorporated into the analysis. Results: This work aims to identify genes that show different gene expression profiles across time. We propose a statistical procedure to discover gene groups with similar profiles using a nonparametric representation that accounts for the autocorrelation in the data. In particular, we first represent each profile in terms of a Fourier basis, and then we screen out genes that are not differentially expressed based on the Fourier coefficients. Finally, we cluster the remaining gene profiles using a model-based approach in the Fourier domain. We evaluate the screening results in terms of sensitivity, specificity, FDR and FNR, compare with the Gaussian process regression screening in a simulation study and illustrate the results by application to yeast cell-cycle microarray expression data with alpha-factor synchronization.The key elements of the proposed methodology: (i) representation of gene profiles in the Fourier domain; (ii) automatic screening of genes based on the Fourier coefficients and taking into account autocorrelation in the data, while controlling the false discovery rate (FDR); (iii) model-based clustering of the remaining gene profiles. Conclusions: Using this method, we identified a set of cell-cycle-regulated time-course yeast genes. The proposed method is general and can be potentially used to identify genes which have the same patterns or biological processes, and help facing the present and forthcoming challenges of data analysis in functional genomics.
High variance in reproductive success generates a false signature of a genetic bottleneck in populations of constant size: a simulation study [BMC Bioinformatics - Latest Articles] 2013-10-15 20:00
Background: Demographic bottlenecks can severely reduce the genetic variation of a population or a species. Establishing whether low genetic variation is caused by a bottleneck or a constantly low effective number of individuals is important to understand a species’ ecology and evolution, and it has implications for conservation management. Recent studies have evaluated the power of several statistical methods developed to identify bottlenecks. However, the false positive rate, i.e. the rate with which a bottleneck signal is misidentified in demographically stable populations, has received little attention. We analyse this type of error (type I) in forward computer simulations of stable populations having greater than Poisson variance in reproductive success (i.e., variance in family sizes). The assumption of Poisson variance underlies bottleneck tests, yet it is commonly violated in species with high fecundity. Results: With large variance in reproductive success (V k ≥ 40, corresponding to a ratio between effective and census size smaller than 0.1), tests based on allele frequencies, allelic sizes, and DNA sequence polymorphisms (heterozygosity excess, M-ratio, and Tajima’s D test) tend to show erroneous signals of a bottleneck. Similarly, strong evidence of population decline is erroneously detected when ancestral and current population sizes are estimated with the model based method MSVAR. Conclusions: Our results suggest caution when interpreting the results of bottleneck tests in species showing high variance in reproductive success. Particularly in species with high fecundity, computer simulations are recommended to confirm the occurrence of a population bottleneck.
Dynamical modelling of phenotypes in a genome-wide RNAi live-cell imaging assay [BMC Bioinformatics - Latest Articles] 2013-10-15 20:00
Background: The combination of time-lapse imaging of live cells with high-throughput perturbation assays is a powerful tool for genetics and cell biology. The Mitocheck project employed this technique to associate thousands of genes with transient biological phenotypes in cell division, cell death and migration. The original analysis of these data proceeded by assigning nuclear morphologies to cells at each time-point using automated image classification, followed by description of population frequencies and temporal distribution of cellular states through event-order maps. One of the choices made by that analysis was not to rely on temporal tracking of the individual cells, due to the relatively low image sampling frequency, and to focus on effects that could be discerned from population-levelbehaviour. Results: Here, we present a variation of this approach that employs explicit modelling by dynamic differential equations of the cellular state populations. Model fitting to the time course data allowed reliable estimation of the penetrance and time of appearance of four types of disruption of the cell cycle: quiescence, mitotic arrest, polynucleation and cell death. Model parameters yielded estimates of the duration of the interphase and mitosis phases. We identified 2190 siRNAs that induced a disruption of the cell cycle at reproducible times, or increased the durations of the interphase or mitosis phases. Conclusions: We quantified the dynamic effects of the siRNAs and compiled them as a resource that can be used to characterize the role of their target genes in cell death, mitosis and cell cycle regulation. The described population-based modelling method might be applicable to other large-scale cell-based assays with temporal readout when only population-level measures are available.
Jimena: efficient computing and system state identification for genetic regulatory networks [BMC Bioinformatics - Latest Articles] 2013-10-10 20:00
Background: Boolean networks capture switching behavior of many naturally occurring regulatory networks. For semi-quantitative modeling, interpolation between ON and OFF states is necessary. The high degree polynomial interpolation of Boolean genetic regulatory networks (GRNs) in cellular processes such as apoptosis or proliferation allows for the modeling of a wider range of node interactions than continuous activator-inhibitor models, but suffers from scaling problems for networks which contain nodes with more than ~10 inputs. Many GRNs from literature or new gene expression experiments exceed those limitations and a new approach was developed. Results: (i) As a part of our new GRN simulation framework Jimena we introduce and setup Boolean-tree-based data structures; (ii) corresponding algorithms greatly expedite the calculation of the polynomial interpolation in almost all cases, thereby expanding the range of networks which can be simulated by this model in reasonable time. (iii) Stable states for discrete models are efficiently counted and identified using binary decision diagrams. As application example, we show how system states can now be sampled efficiently in small up to large scale hormone disease networks (Arabidopsis thaliana development and immunity, pathogen Pseudomonas syringae and modulation by cytokinins and plant hormones). Conclusions: Jimena simulates currently available GRNs about 10-100 times faster than the previous implementation of the polynomial interpolation model and even greater gains are achieved for large scale-free networks. This speed-up also facilitates a much more thorough sampling of continuous state spaces which may lead to the identification of new stable states. Mutants of large networks can be constructed and analyzed very quickly enabling new insights into network robustness and behavior.
cudaMap: a GPU accelerated program for gene expression connectivity mapping [BMC Bioinformatics - Latest Articles] 2013-10-10 20:00
Background: Modern cancer research often involves large datasets and the use of sophisticated statistical techniques. Together these add a heavy computational load to the analysis, which is often coupled with issues surrounding data accessibility. Connectivity mapping is an advanced bioinformatic and computational technique dedicated to therapeutics discovery and drug re-purposing around differential gene expression analysis. On a normal desktop PC, it is common for the connectivity mapping task with a single gene signature to take > 2h to complete using sscMap, a popular Java application that runs on standard CPUs (Central Processing Units). Here, we describe new software, cudaMap, which has been implemented using CUDA C/C++ to harness the computational power of NVIDIA GPUs (Graphics Processing Units) to greatly reduce processing times for connectivity mapping. Results: cudaMap can identify candidate therapeutics from the same signature in just over thirty seconds when using an NVIDIA Tesla C2050 GPU. Results from the analysis of multiple gene signatures, which would previously have taken several days, can now be obtained in as little as 10 minutes, greatly facilitating candidate therapeutics discovery with high throughput. We are able to demonstrate dramatic speed differentials between GPU assisted performance and CPU executions as the computational load increases for high accuracy evaluation of statistical significance. Conclusion: Emerging ‘omics’ technologies are constantly increasing the volume of data and information to be processed in all areas of biomedical research. Embracing the multicore functionality of GPUs represents a major avenue of local accelerated computing. cudaMap will make a strong contribution in the discovery of candidate therapeutics by enabling speedy execution of heavy duty connectivity mapping tasks, which are increasingly required in modern cancer research. cudaMap is open source and can be freely downloaded from http://purl.oclc.org/NET/cudaMap.
Lipid exposure prediction enhances the inference of rotational angles of transmembrane helices [BMC Bioinformatics - Latest Articles] 2013-10-10 20:00
Background: Since membrane protein structures are challenging to crystallize, computational approaches are essential for elucidating the sequence-to-structure relationships. Structural modeling of membrane proteins requires a multidimensional approach, and one critical geometric parameter is the rotational angle of transmembrane helices. Rotational angles of transmembrane helices are characterized by their folded structures and could be inferred by the hydrophobic moment; however, the folding mechanism of membrane proteins is not yet fully understood. The rotational angle of a transmembrane helix is related to the exposed surface of a transmembrane helix, since lipid exposure gives the degree of accessibility of each residue in lipid environment. To the best of our knowledge, there have been few advances in investigating whether an environment descriptor of lipid exposure could infer a geometric parameter of rotational angle. Results: Here, we present an analysis of the relationship between rotational angles and lipid exposure and a support-vector-machine method, called TMexpo, for predicting both structural features from sequences. First, we observed from the development set of 89 protein chains that the lipid exposure, i.e., the relative accessible surface area (rASA) of residues in the lipid environment, generated from high-resolution protein structures could infer the rotational angles with a mean absolute angular error (MAAE) of 46.32˚. More importantly, the predicted rASA from TMexpo achieved an MAAE of 51.05˚, which is better than 71.47˚ obtained by the best of the compared hydrophobicity scales. Lastly, TMexpo outperformed the compared methods in rASA prediction on the independent test set of 21 protein chains and achieved an overall Matthew’s correlation coefficient, accuracy, sensitivity, specificity, and precision of 0.51, 75.26%, 81.30%, 69.15%, and 72.73%, respectively. TMexpo is publicly available at http://bio-cluster.iis.sinica.edu.tw/TMexpo. Conclusions: TMexpo can better predict rASA and rotational angles than the compared methods. When rotational angles can be accurately predicted, free modeling of transmembrane protein structures in turn may benefit from a reduced complexity in ensembles with a significantly less number of packing arrangements. Furthermore, sequence-based prediction of both rotational angle and lipid exposure can provide essential information when high-resolution structures are unavailable and contribute to experimental design to elucidate transmembrane protein functions.
Inferring Polymorphism-Induced Regulatory Gene Networks Active in Human Lymphocyte Cell Lines by Weighted Linear Mixed Model Analysis of Multiple RNA-Seq Datasets [PLOS ONE] 2013-10-30 17:00
by Wensheng Zhang, Andrea Edwards, Erik K. Flemington, Kun Zhang
Single-nucleotide polymorphisms (SNPs) contribute to the between-individual expression variation of many genes. A regulatory (trait-associated) SNP is usually located near or within a (host) gene, possibly influencing the gene’s transcription or/and post-transcriptional modification. But its targets may also include genes that are physically farther away from it. A heuristic explanation of such multiple-target interferences is that the host gene transfers the SNP genotypic effects to the distant gene(s) by a transcriptional or signaling cascade. These connections between the host genes (regulators) and the distant genes (targets) make the genetic analysis of gene expression traits a promising approach for identifying unknown regulatory relationships. In this study, through a mixed model analysis of multi-source digital expression profiling for 140 human lymphocyte cell lines (LCLs) and the genotypes distributed by the international HapMap project, we identified 45 thousands of potential SNP-induced regulatory relationships among genes (the significance level for the underlying associations between expression traits and SNP genotypes was set at FDR < 0.01). We grouped the identified relationships into four classes (paradigms) according to the two different mechanisms by which the regulatory SNPs affect their cis- and trans- regulated genes, modifying mRNA level or altering transcript splicing patterns. We further organized the relationships in each class into a set of network modules with the cis- regulated genes as hubs. We found that the target genes in a network module were often characterized by significant functional similarity, and the distributions of the target genes in three out of the four networks roughly resemble a power-law, a typical pattern of gene networks obtained from mutation experiments. By two case studies, we also demonstrated that significant biological insights can be inferred from the identified network modules.Talaromyces columbinus sp. nov., and Genealogical Concordance Analysis in Talaromyces Clade 2a [PLOS ONE] 2013-10-30 17:00
by Stephen W. Peterson, Željko Jurjević
During the course of mold surveys, a set of Talaromyces isolates were obtained that did not fit any described species. Phenotypic examination of these isolates showed that they were similar to T. piceus but differed in some growth characteristics. Multilocus DNA sequence data were obtained for the new isolates and some related species in the broader, more inclusive clade, and the data were analyzed using genealogical concordance. The new isolates are described as Talaromyces columbinus. From analysis of the related species, Penicillium rugulosum var. atricolum is given species status in Talaromyces as T. atricola. Penicillium tardum and P. chrysitis were showed to be synonyms of T. rugulosus. Penicillium scorteum and T. phialosporus were showed to be conspecific and under the rule of priority T. scorteus is the proper name for isolates previously known as T. phialosporus. Talaromyces wortmanii was showed to be distinct from Penicillium concavorugulosum and T. variabilis but the relationship of the latter two species remains unresolved. Examination of ITS sequences from GenBank showed that T. columbinus has previously been reported from human lung infections under the name Penicillium piceum.A One-Degree-of-Freedom Test for Supra-Multiplicativity of SNP Effects [PLOS ONE] 2013-10-30 17:00
by Christine Herold, Alfredo Ramirez, Dmitriy Drichel, André Lacour, Tatsiana Vaitsiakhovich, Markus M. Nöthen, Frank Jessen, Wolfgang Maier, Tim Becker
Deviation from multiplicativity of genetic risk factors is biologically plausible and might explain why Genome-wide association studies (GWAS) so far could unravel only a portion of disease heritability. Still, evidence for SNP-SNP epistasis has rarely been reported, suggesting that 2-SNP models are overly simplistic. In this context, it was recently proposed that the genetic architecture of complex diseases could follow limiting pathway models. These models are defined by a critical risk allele load and imply multiple high-dimensional interactions. Here, we present a computationally efficient one-degree-of-freedom “supra-multiplicativity-test” (SMT) for SNP sets of size 2 to 500 that is designed to detect risk alleles whose joint effect is fortified when they occur together in the same individual. Via a simulation study we show that the SMT is powerful in the presence of threshold models, even when only about 30–45% of the model SNPs are available. In addition, we demonstrate that the SMT outperforms standard interaction analysis under recessive models involving just a few SNPs. We apply our test to 10 consensus Alzheimer’s disease (AD) susceptibility SNPs that were previously identified by GWAS and obtain evidence for supra-multiplicativity () that is not attributable to either two-way or three-way interaction.CanDrA: Cancer-Specific Driver Missense Mutation Annotation with Optimized Features [PLOS ONE] 2013-10-30 17:00
by Yong Mao, Han Chen, Han Liang, Funda Meric-Bernstam, Gordon B. Mills, Ken Chen
Driver mutations are somatic mutations that provide growth advantage to tumor cells, while passenger mutations are those not functionally related to oncogenesis. Distinguishing drivers from passengers is challenging because drivers occur much less frequently than passengers, they tend to have low prevalence, their functions are multifactorial and not intuitively obvious. Missense mutations are excellent candidates as drivers, as they occur more frequently and are potentially easier to identify than other types of mutations. Although several methods have been developed for predicting the functional impact of missense mutations, only a few have been specifically designed for identifying driver mutations. As more mutations are being discovered, more accurate predictive models can be developed using machine learning approaches that systematically characterize the commonality and peculiarity of missense mutations under the background of specific cancer types. Here, we present a cancer driver annotation (CanDrA) tool that predicts missense driver mutations based on a set of 95 structural and evolutionary features computed by over 10 functional prediction algorithms such as CHASM, SIFT, and MutationAssessor. Through feature optimization and supervised training, CanDrA outperforms existing tools in analyzing the glioblastoma multiforme and ovarian carcinoma data sets in The Cancer Genome Atlas and the Cancer Cell Line Encyclopedia project.Extension of Life Span by Impaired Glucose Metabolism in Caenorhabditis elegans Is Accompanied by Structural Rearrangements of the Transcriptomic Network [PLOS ONE] 2013-10-30 17:00
by Steffen Priebe, Uwe Menzel, Kim Zarse, Marco Groth, Matthias Platzer, Michael Ristow, Reinhard Guthke
Glucose restriction mimicked by feeding the roundworm Caenorhabditis elegans with 2-deoxy-D-glucose (DOG) - a glucose molecule that lacks the ability to undergo glycolysis - has been found to increase the life span of the nematodes considerably. To facilitate understanding of the molecular mechanisms behind this life extension, we analyzed transcriptomes of DOG-treated and untreated roundworms obtained by RNA-seq at different ages. We found that, depending on age, DOG changes the magnitude of the expression values of about 2 to 24 percent of the genes significantly, although our results reveal that the gross changes introduced by DOG are small compared to the age-induced changes. We found that 27 genes are constantly either up- or down-regulated by DOG over the whole life span, among them several members of the cytochrome P450 family. The monotonic change with age of the temporal expression patterns of the genes was investigated, leading to the result that 21 genes reverse their monotonic behaviour under impaired glycolysis. Put simply, the DOG-treatment reduces the gross transcriptional activity but increases the interconnectedness of gene expression. However, a detailed analysis of network parameters discloses that the introduced changes differ remarkably between individual signalling pathways. We found a reorganization of the hubs of the mTOR pathway when standard diet is replaced by DOG feeding. By constructing correlation based difference networks, we identified those signalling pathways that are most vigorously changed by impaired glycolysis. Taken together, we have found a number of genes and pathways that are potentially involved in the DOG-driven extension of life span of C. elegans. Furthermore, our results demonstrate how the network structure of ageing-relevant signalling pathways is reorganised under impaired glycolysis.Fibroblast Growth Factor Receptors as Novel Therapeutic Targets in SNF5-Deleted Malignant Rhabdoid Tumors [PLOS ONE] 2013-10-30 17:00
by Simon Wöhrle, Andreas Weiss, Moriko Ito, Audrey Kauffmann, Masato Murakami, Zainab Jagani, Anne Thuery, Beatrice Bauer-Probst, Flavia Reimann, Christelle Stamm, Astrid Pornon, Vincent Romanet, Vito Guagnano, Thomas Brümmendorf, William R. Sellers, Francesco Hofmann, Charles W. M. Roberts, Diana Graus Porta
Malignant rhabdoid tumors (MRTs) are aggressive pediatric cancers arising in brain, kidney and soft tissues, which are characterized by loss of the tumor suppressor SNF5/SMARCB1. MRTs are poorly responsive to chemotherapy and thus a high unmet clinical need exists for novel therapies for MRT patients. SNF5 is a core subunit of the SWI/SNF chromatin remodeling complex which affects gene expression by nucleosome remodeling. Here, we report that loss of SNF5 function correlates with increased expression of fibroblast growth factor receptors (FGFRs) in MRT cell lines and primary tumors and that re-expression of SNF5 in MRT cells causes a marked repression of FGFR expression. Conversely, siRNA-mediated impairment of SWI/SNF function leads to elevated levels of FGFR2 in human fibroblasts. In vivo, treatment with NVP-BGJ398, a selective FGFR inhibitor, blocks progression of a murine MRT model. Hence, we identify FGFR signaling as an aberrantly activated oncogenic pathway in MRTs and propose pharmacological inhibition of FGFRs as a potential novel clinical therapy for MRTs.Metabolic Proximity in the Order of Colonization of a Microbial Community [PLOS ONE] 2013-10-30 17:00
by Varun Mazumdar, Salomon Amar, Daniel Segrè
Microbial biofilms are often composed of multiple bacterial species that accumulate by adhering to a surface and to each other. Biofilms can be resistant to antibiotics and physical stresses, posing unresolved challenges in the fight against infectious diseases. It has been suggested that early colonizers of certain biofilms could cause local environmental changes, favoring the aggregation of subsequent organisms. Here we ask whether the enzyme content of different microbes in a well-characterized dental biofilm can be used to predict their order of colonization. We define a metabolic distance between different species, based on the overlap in their enzyme content. We next use this metric to quantify the average metabolic distance between neighboring organisms in the biofilm. We find that this distance is significantly smaller than the one observed for a random choice of prokaryotes, probably reflecting the environmental constraints on metabolic function of the community. More surprisingly, this metabolic metric is able to discriminate between observed and randomized orders of colonization of the biofilm, with the observed orders displaying smaller metabolic distance than randomized ones. By complementing these results with the analysis of individual vs. joint metabolic networks, we find that the tendency towards minimal metabolic distance may be counter-balanced by a propensity to pair organisms with maximal joint potential for synergistic interactions. The trade-off between these two tendencies may create a “sweet spot” of optimal inter-organism distance, with possible broad implications for our understanding of microbial community organization.RNA-Sequencing Analysis of TCDD-Induced Responses in Zebrafish Liver Reveals High Relatedness to In Vivo Mammalian Models and Conserved Biological Pathways [PLOS ONE] 2013-10-30 17:00
by Zhi-Hua Li, Hongyan Xu, Weiling Zheng, Siew Hong Lam, Zhiyuan Gong
TCDD is one of the most persistent environmental toxicants in biological systems and its effect through aryl hydrocarbon receptor (AhR) has been well characterized. However, the information on TCDD-induced toxicity in other molecular pathways is rather limited. To fully understand molecular toxicity of TCDD in an in vivo animal model, adult zebrafish were exposed to TCDD at 10 nM for 96 h and the livers were sampled for RNA-sequencing based transcriptomic profiling. A total of 1,058 differently expressed genes were identified based on fold-change>2 and TPM (transcripts per million) >10. Among the top 20 up-regulated genes, 10 novel responsive genes were identified and verified by RT-qPCR analysis on independent samples. Transcriptomic analysis indicated several deregulated pathways associated with cell cycle, endocrine disruptors, signal transduction and immune systems. Comparative analyses of TCDD-induced transcriptomic changes between fish and mammalian models revealed that proteomic pathway is consistently up-regulated while calcium signaling pathway and several immune-related pathways are generally down-regulated. Finally, our study also suggested that zebrafish model showed greater similarity to in vivo mammalian models than in vitro models. Our study indicated that the zebrafish is a valuable in vivo model in toxicogenomic analyses for understanding molecular toxicity of environmental toxicants relevant to human health. The expression profiles associated with TCDD could be useful for monitoring environmental dioxin and dioxin-like contamination.Evaluation of Alignment Algorithms for Discovery and Identification of Pathogens Using RNA-Seq [PLOS ONE] 2013-10-30 17:00
by Ivan Borozan, Stuart N. Watt, Vincent Ferretti
Next-generation sequencing technologies provide an unparallelled opportunity for the characterization and discovery of known and novel viruses. Because viruses are known to have the highest mutation rates when compared to eukaryotic and bacterial organisms, we assess the extent to which eleven well-known alignment algorithms (BLAST, BLAT, BWA, BWA-SW, BWA-MEM, BFAST, Bowtie2, Novoalign, GSNAP, SHRiMP2 and STAR) can be used for characterizing mutated and non-mutated viral sequences - including those that exhibit RNA splicing - in transcriptome samples. To evaluate aligners objectively we developed a realistic RNA-Seq simulation and evaluation framework (RiSER) and propose a new combined score to rank aligners for viral characterization in terms of their precision, sensitivity and alignment accuracy. We used RiSER to simulate both human and viral read sequences and suggest the best set of aligners for viral sequence characterization in human transcriptome samples. Our results show that significant and substantial differences exist between aligners and that a digital-subtraction-based viral identification framework can and should use different aligners for different parts of the process. We determine the extent to which mutated viral sequences can be effectively characterized and show that more sensitive aligners such as BLAST, BFAST, SHRiMP2, BWA-SW and GSNAP can accurately characterize substantially divergent viral sequences with up to 15% overall sequence mutation rate. We believe that the results presented here will be useful to researchers choosing aligners for viral sequence characterization using next-generation sequencing data.Evidence of Sympatry of Clade A and Clade B Head Lice in a Pre-Columbian Chilean Mummy from Camarones [PLOS ONE] 2013-10-30 17:00
by Amina Boutellis, Rezak Drali, Mario A. Rivera, Kosta Y. Mumcuoglu, Didier Raoult
Three different lineages of head lice are known to parasitize humans. Clade A, which is currently worldwide in distribution, was previously demonstrated to be present in the Americas before the time of Columbus. The two other types of head lice are geographically restricted to America and Australia for clade B and to Africa and Asia for clade C. In this study, we tested two operculated nits from a 4,000-year-old Chilean mummy of Camarones for the presence of the partial Cytb mitochondrial gene (270 bp). Our finding shows that clade B head lice were present in America before the arrival of the European colonists.In This Issue [This Week in PNAS] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Methane emissions associated with natural gas production in the United States Natural gas well in Texas. ©iStockphoto/sdphotography. Significant uncertainty surrounds the levels of methane emissions associated with natural gas production in the United States. National estimates of methane emissions from natural gas production have generally relied on engineering models and...
No evidence for manganese-oxidizing photosynthesis [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
In PNAS, Johnson et al. (1) argue that a hitherto unknown form of photosynthesis using Mn as electron donor was the precursor to the evolution of cyanobacteria. The authors conclude this from chemical tracers, particularly putative remnants of Mn oxides in the rock record some 2.4 billion y ago (1)....
Reply to Jones and Crowe [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Jones and Crowe (1) raise issues already addressed in our article (2) based on an inaccurate grasp of the literature and several logical misconceptions. The authors suggest that inputs we chose in our kinetic calculations are unsuitable because we used values only from the Black Sea. As described, we made...
Double layer in ionic liquids [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
According to a recent article in PNAS by Gebbie et al., ionic liquids (ILs) should be considered as dilute electrolyte solutions, consisting of ion pairs of which only a small fraction are dissociated (1). This claim is based upon measurements of weak attractive forces between a mica surface and a...
On diffuse double layers in a common ionic liquid [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Perkin et al. assert that ionic liquids (ILs) should behave as strongly dissociated (uncorrelated) electrolytes and use this premise to question our approach of implementing the Derjaguin–Landau–Verwey–Overbeek (DLVO) theory in the analysis of our experimental observations (1). Perkin et al. also assert that the long-range attraction that we observed is...
End-of-life electrical surges [Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
We read the study by Borjigin et al. about postcardiac arrest electrical activity and the possible explanation for near death experiences (NDEs) with great interest (1). Our research group at The George Washington University Medical Center published an observation of end-of-life electrical surges (ELES) in 2009 (2). In that study,...
Limits of BIS scoring in patients at near death [Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
We concur with Chawla and Seneff (1) on the importance of gaining scientific understanding of the dying process (2) and are aware of their interesting study demonstrating a brief surge of electrical activity in dying human patients (3). Chawla and Seneff (1) state that our study “largely confirms” their hypothesis...
Retrospective: Tony Pawson [Retrospectives] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
It is with great sorrow that I write that Tony Pawson, a good friend, great colleague, generous collaborator, and brilliant scientist, died at the age of 60 on August 7, 2013. Tony was a trail-blazer and innovator in his studies of the mechanisms underlying intracellular signaling. His work had an...
Live fast, die young, win the sperm competition [Ecology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Consider the two mammalian species shown in Fig. 1. The brown antechinus (Antechinus stuartii) has a fast life cycle and “big bang” reproduction, with males that mate for only a single breeding season in 1 y and then die a programmed death associated with lethal immune system collapse (1). The...
Breaking the limits of artificial ubiquitination [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Neurodegenerative diseases such as Parkinson, Alzheimer’s, and Huntington are proteopathic disorders characterized by the accumulation of intracellular insoluble aggregates of misfolded proteins and are one of the major medical concerns of our modern society. Particularly, Parkinson disease is a synucleinopathy caused by the cytosolic aggregation of α-Synuclein into Lewy bodies...
Antibody-directed stem cell differentiation [Applied Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Stem cells are highly specialized cells endowed with unlimited replicative self-renewing potential. These cells are capable of either limited multipotent (adult stem cells, ASC) or unlimited pluripotent (embryonic stem cells, ESC) differentiation to somatic cell lineages. Control of this differentiation process holds great promise in areas such as tissue regeneration...
Nuclear translation for immunosurveillance [Immunology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Murphy’s Law, “anything that can happen, will happen,” constitutes a reasonable working philosophy for biologists. A corollary is that seemingly minor processes can have major consequences. In exploring how the immune system monitors cells for abnormal gene expression, Apcher et al. (1) provide solid evidence supporting two highly controversial processes:...
Structural responses of gap junctions to AB5 toxin [Cell Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Gap junctions (GJs) represent connexin-rich membrane domains that connect interiors of adjoining cells in mammalian tissues. How fast GJs can respond to bacterial pathogens has not been known previously. Using Bessel beam plane illumination and confocal spinning disk microscopy, we found fast (∼500 ms) formation of connexin-depleted regions (CDRs) inside...
CIN rate predicts effect of aneuploidy on tumors [Cell Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Aneuploidy, a chromosome content other than a multiple of the haploid number, is a common feature of cancer cells. Whole chromosomal aneuploidy accompanying ongoing chromosomal instability in mice resulting from reduced levels of the centromere-linked motor protein CENP-E has been reported to increase the incidence of spleen and lung tumors,...
NO regulates activity-dependent synapse formation [Neuroscience] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Learning related paradigms play an important role in shaping the development and specificity of synaptic networks, notably by regulating mechanisms of spine growth and pruning. The molecular events underlying these synaptic rearrangements remain poorly understood. Here we identify NO signaling as a key mediator of activity-dependent excitatory synapse development. We...
TBI in flies [Neuroscience] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Traumatic brain injury (TBI) is a substantial health issue worldwide, yet the mechanisms responsible for its complex spectrum of pathologies remains largely unknown. To investigate the mechanisms underlying TBI pathologies, we developed a model of TBI in Drosophila melanogaster. The model allows us to take advantage of the wealth of...
Drug resistance in prion therapeutics [Pharmacology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt–Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times...
Population neuroscience [Social Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The last decades of neuroscience research have produced immense progress in the methods available to understand brain structure and function. Social, cognitive, clinical, affective, economic, communication, and developmental neurosciences have begun to map the relationships between neuro-psychological processes and behavioral outcomes, yielding a new understanding of human behavior and promising...
Mass-independent fractionation concepts [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Key experimental and theoretical features of mass-independent fractionation (MIF) of isotopes, also known as the η-effect, are summarized, including its difference from the exit channel zero-point energy difference effect. The latter exactly cancels in the MIF. One key experimental result is that the MIF for O3 formation is a low-pressure...
DWA1 controls drought-induced wax deposition [Agricultural Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Drought stress is a major limiting factor for crop production. Cuticular wax plays an important role in preventing water loss from drought stress. However, the genetic control of cuticular wax deposition under drought stress conditions has not been characterized. Here, we identified a rice gene Drought-Induced Wax Accumulation 1 (DWA1)...
Bispecific antibody targeting prostate cancer [Applied Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative...
Functional antibody selection in ES cells [Applied Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Antibodies that modulate receptor function have great untapped potential in the control of stem cell differentiation. In contrast to many natural ligands, antibodies are stable, exquisitely specific, and are unaffected by the regulatory mechanisms that act on natural ligands. Here we describe an innovative system for identifying such antibodies by...
Fatty acid saturation by microorganisms [Applied Biological Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed...
Water's second glass transition [Applied Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The glassy states of water are of common interest as the majority of H2O in space is in the glassy state and especially because a proper description of this phenomenon is considered to be the key to our understanding why liquid water shows exceptional properties, different from all other liquids....
Chemical biology of polyubiquitinated {alpha}-Synuclein [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Ubiquitination regulates, via different modes of modifications, a variety of biological processes, and aberrations in the process have been implicated in the pathogenesis of several neurodegenerative diseases. However, our ability to dissect the pathophysiological relevance of the ubiquitination code has been hampered due to the lack of methods that allow...
Crystal structure of Bacillus subtilis GabR [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Bacillus subtilis GabR is a transcription factor that regulates gamma-aminobutyric acid (GABA) metabolism. GabR is a member of the understudied MocR/GabR subfamily of the GntR family of transcription regulators. A typical MocR/GabR-type regulator is a chimeric protein containing a short N-terminal helix-turn-helix DNA-binding domain and a long C-terminal pyridoxal 5′-phosphate...
Modulating transcription by splicing & DNA signals [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation, and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the...
Structure of KIT inhibitor antibodies [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Somatic oncogenic mutations in the receptor tyrosine kinase KIT function as major drivers of gastrointestinal stromal tumors and a subset of acute myeloid leukemia, melanoma, and other cancers. Although treatment of these cancers with tyrosine kinase inhibitors shows dramatic responses and durable disease control, drug resistance followed by clinical progression...
Inhibition of prothrombinase by TFPI{alpha} [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Tissue factor (TF) pathway inhibitor (TFPI) is a well-characterized activated factor X (FXa)-dependent inhibitor of TF-initiated coagulation produced in two alternatively spliced isoforms, TFPIα and TFPIβ. The TFPIα C terminus has a basic sequence nearly identical to a portion of the factor V (FV) B domain necessary for maintaining FV...
Role for Ape1 in telomere protection [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The major mammalian apurinic/apyrimidinic endonuclease Ape1 is a multifunctional protein operating in protection of cells from oxidative stress via its DNA repair, redox, and transcription regulatory activities. The importance of Ape1 has been marked by previous work demonstrating its requirement for viability in mammalian cells. However, beyond a requirement for...
Kinetic model hAgo2 [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Argonaute (Ago) proteins are the key component of the RNA-induced silencing complex and mediate RNA interference (RNAi) in association with small RNAs. Although overall the mechanism of RNAi is well understood, many molecular details of this complex process are not. Here we report about in-depth steady-state and, in particular, pre-steady-state...
In vitro reconstitution of cellulose biosynthesis [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Cellulose is a linear extracellular polysaccharide. It is synthesized by membrane-embedded glycosyltransferases that processively polymerize UDP-activated glucose. Polymer synthesis is coupled to membrane translocation through a channel formed by the cellulose synthase. Although eukaryotic cellulose synthases function in macromolecular complexes containing several different enzyme isoforms, prokaryotic synthases associate with additional...
Crystal structure of a glucose transporter [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H+ symporter in an...
Archaeal oligosaccharyltransferase structure [Biochemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Oligosaccharyltransferase transfers an oligosaccharide chain to the asparagine residues in proteins. The archaeal and eubacterial oligosaccharyltransferases are single subunit membrane enzymes, referred to as “AglB” (archaeal glycosylation B) and “PglB” (protein glycosylation B), respectively. Only one crystal structure of a full-length PglB has been solved. Here we report the crystal...
Ion occupancy and inactivation in K+ channels [Biophysics and Computational Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
K+ channels distinguish K+ from Na+ in the selectivity filter, which consists of four ion-binding sites (S1–S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a...
MiR-26a targets TET enzymes [Cell Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Ten eleven translocation (TET) enzymes (TET1/TET2/TET3) and thymine DNA glycosylase (TDG) play crucial roles in early embryonic and germ cell development by mediating DNA demethylation. However, the molecular mechanisms that regulate TETs/TDG expression and their role in cellular differentiation, including that of the pancreas, are not known. Here, we report...
Regulation of DHHC9 by miR-134 in interneurons [Cell Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
MicroRNA-134 (miR-134) serves as a widely accepted model for microRNA function in synaptic plasticity. In this model, synaptic activity stimulates miR-134 expression, which then regulates dendrite growth and spine formation. By using a ratiometric microRNA sensor, we found, unexpectedly, that miR-134 activity in cortical neurons was restricted to interneurons. Using...
Sulfur MIF in photoexcitation of SO2 [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Signatures of mass-independent isotope fractionation (MIF) are found in the oxygen (16O,17O,18O) and sulfur (32S, 33S, 34S, 36S) isotope systems and serve as important tracers of past and present atmospheric processes. These unique isotope signatures signify the breakdown of the traditional theory of isotope fractionation, but the physical chemistry of...
Nonenzymatic copying of synthetic genetic polymer [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis...
Steric effects in molecule-surface collisions [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Molecules typically must point in specific relative directions to participate efficiently in energy transfer and reactions. For example, Förster energy transfer favors specific relative directions of each molecule’s transition dipole [Förster T (1948) Ann Phys 2(1-2):55–75] and electron transfer between gas-phase molecules often depends on the relative orientation of orbitals...
Dynamic assembly of nanomotors [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Nano- and microscale motors powered by catalytic reactions exhibit collective behavior such as swarming, predator–prey interactions, and chemotaxis that resemble those of biological microorganisms. A quantitative understanding of the catalytically generated forces between particles that lead to these behaviors has so far been lacking. Observations and numerical simulations of pairwise...
MDMX autoinhibition [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
MDM2 and MDMX are homologous proteins that bind to p53 and regulate its activity. Both contain three folded domains and ∼70% intrinsically disordered regions. Previous detailed structural and biophysical studies have concentrated on the isolated folded domains. The N-terminal domains of both exhibit high affinity for the disordered N-terminal of...
Nonnative contacts and protein folding mechanism [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The recent availability of long equilibrium simulations of protein folding in atomistic detail for more than 10 proteins allows us to identify the key interactions driving folding. We find that the collective fraction of native amino acid contacts, Q, captures remarkably well the transition states for all the proteins with...
Transition path distributions in protein folding [Chemistry] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Advances in computing have enabled microsecond all-atom molecular dynamics trajectories of protein folding that can be used to compare with and test critical assumptions of theoretical models. We show that recent simulations by the Shaw group (10, 11, 14, 15) are consistent with a key assumption of an Ising-like theoretical...
Mass independent isotope effects [Physical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Stable isotope ratio variations are regulated by physical and chemical laws. These rules depend on a relation with mass differences between isotopes. New classes of isotope variation effects that deviate from mass dependent laws, termed mass independent isotope effects, were discovered in 1983 and have a wide range of applications...
Role of polysulfide in Neoarchean pyrite formation [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
It is generally thought that the sulfate reduction metabolism is ancient and would have been established well before the Neoarchean. It is puzzling, therefore, that the sulfur isotope record of the Neoarchean is characterized by a signal of atmospheric mass-independent chemistry rather than a strong overprint by sulfate reducers. Here,...
Marine sulfur cycle constraints on S MIF [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Mass-independent fractionation of sulfur isotopes (S MIF) in Archean and Paleoproterozoic rocks provides strong evidence for an anoxic atmosphere before ∼2,400 Ma. However, the origin of this isotopic anomaly remains unclear, as does the identity of the molecules that carried it from the atmosphere to Earth’s surface. Irrespective of the...
S-isotopic fractionation in VUV photolysis of H2S [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Select meteoritic classes possess mass-independent sulfur isotopic compositions in sulfide and organic phases. Photochemistry in the solar nebula has been attributed as a source of these anomalies. Hydrogen sulfide (H2S) is the most abundant gas-phase species in the solar nebula, and hence, photodissociation of H2S by solar vacuum UV (VUV)...
MIF via SO2 photoexcitation [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Natural climate variation, such as that caused by volcanoes, is the basis for identifying anthropogenic climate change. However, knowledge of the history of volcanic activity is inadequate, particularly concerning the explosivity of specific events. Some material is deposited in ice cores, but the concentration of glacial sulfate does not distinguish...
Oxygen isotope anomaly of sulfate aerosol and ENSO [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The ability of sulfate aerosols to reflect solar radiation and simultaneously act as cloud condensation nuclei renders them central players in the global climate system. The oxidation of S(IV) compounds and their transport as stable S(VI) in the Earth’s system are intricately linked to planetary scale processes, and precise characterization...
Nitrate isotopologues in a tropical MBL [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Long-term observations of the reactive chemical composition of the tropical marine boundary layer (MBL) are rare, despite its crucial role for the chemical stability of the atmosphere. Recent observations of reactive bromine species in the tropical MBL showed unexpectedly high levels that could potentially have an impact on the ozone...
17O-excess at Vostok indicates stratospheric input [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Combined measurements of water isotopologues of a snow pit at Vostok over the past 60 y reveal a unique signature that cannot be explained only by climatic features as usually done. Comparisons of the data using a general circulation model and a simpler isotopic distillation model reveal a stratospheric signature...
Oxygen isotopic composition of stratospheric CO2 [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
We report observations of stratospheric CO2 that reveal surprisingly large anomalous enrichments in 17O that vary systematically with latitude, altitude, and season. The triple isotope slopes reached 1.95 ± 0.05(1σ) in the middle stratosphere and 2.22 ± 0.07 in the Arctic vortex versus 1.71 ± 0.03 from previous observations and...
Duration of sulfate-17 depletion at 635 Ma [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The ∼635 Ma Marinoan glaciation is marked by dramatic Earth system perturbations. Deposition of nonmass-dependently 17O-depleted sulfate (SO42−) in worldwide postglacial sediments is, thus far, unique to this glaciation. It is proposed that an extremely high-pCO2 atmosphere can result in highly 17O-depleted atmospheric O2, or the Marinoan Oxygen-17 Depletion (MOSD)...
CO2 UV spectrum from first principles [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
We present a first principles study of the carbon dioxide (CO2) photodissociation process in the 150- to 210-nm wavelength range, with emphasis on photolysis below the carbon monoxide + singlet channel threshold at ∼167 nm. The calculations reproduce experimental absorption cross-sections at a resolution of ∼0.5 nm without scaling the...
Mass-independent fractionation in ozone formation [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Theoretical treatment of ozone forming reaction is developed within the framework of mixed quantum/classical dynamics. Formation and stabilization steps of the energy transfer mechanism are both studied, which allows simultaneous capture of the delta zero-point energy effect and η-effect and identification of the molecular level origin of mass-independent isotope fractionation....
Nuclear volume isotope effects in crystals [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Mass-independent isotope fractionations driven by differences in volumes and shapes of nuclei (the field shift effect) are known in several elements and are likely to be found in more. All-electron relativistic electronic structure calculations can predict this effect but at present are computationally intensive and limited to modeling small gas...
Crystallization of Mg-Ca-CO3 at ambient conditions [Earth, Atmospheric, and Planetary Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Dolomite and magnesite are simple anhydrous calcium and/or magnesium carbonate minerals occurring mostly at Earth surfaces. However, laboratory synthesis of neither species at ambient temperature and pressure conditions has been proven practically possible, and the lack of success was assumed to be related to the strong solvation shells of magnesium...
Evolution of suicidal reproduction in mammals [Ecology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Suicidal reproduction (semelparity) has evolved in only four genera of mammals. In these insectivorous marsupials, all males die after mating, when failure of the corticosteroid feedback mechanism elevates stress hormone levels during the mating season and causes lethal immune system collapse (die-off). We quantitatively test and resolve the evolutionary causes...
Dislocation-defect interaction mechanisms map [Engineering] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Probing the mechanisms of defect–defect interactions at strain rates lower than 106 s−1 is an unresolved challenge to date to molecular dynamics (MD) techniques. Here we propose an original atomistic approach based on transition state theory and the concept of a strain-dependent effective activation barrier that is capable of simulating...
Direct transfer of graphene to flexible substrates [Engineering] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
In this paper we explore the direct transfer via lamination of chemical vapor deposition graphene onto different flexible substrates. The transfer method investigated here is fast, simple, and does not require an intermediate transfer membrane, such as polymethylmethacrylate, which needs to be removed afterward. Various substrates of general interest in...
Energy recapture aids propulsive advantage [Engineering] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Gelatinous zooplankton populations are well known for their ability to take over perturbed ecosystems. The ability of these animals to outcompete and functionally replace fish that exhibit an effective visual predatory mode is counterintuitive because jellyfish are described as inefficient swimmers that must rely on direct contact with prey to...
Methane emissions at natural gas production sites [Environmental Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Engineering estimates of methane emissions from natural gas production have led to varied projections of national emissions. This work reports direct measurements of methane emissions at 190 onshore natural gas sites in the United States (150 production sites, 27 well completion flowbacks, 9 well unloadings, and 4 workovers). For well...
Close relatives make bad neighbors [Evolution] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Invasive species have great ecological and economic impacts and are difficult to control once established, making the ability to understand and predict invasive behavior highly desirable. Preemptive measures to prevent potential invasive species from reaching new habitats are the most economically and environmentally efficient form of management. Darwin’s naturalization hypothesis...
Universal exPression Codes [Genetics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Over the past two decades, many biotechnology platforms have been developed for high-throughput gene expression profiling. However, because each platform is subject to technology-specific biases and produces distinct raw-data distributions, researchers have experienced difficulty in integrating data across platforms. Data integration is crucial to data-generating consortiums, researchers transitioning to newer...
Stretch enhancers are linked to common diseases [Genetics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant...
ELL, a novel TFIIH partner [Genetics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
DNA lesions that block transcription may cause cell death even when repaired, if transcription does not restart to reestablish cellular metabolism. However, transcription resumption after individual DNA-lesion repair remains poorly described in mechanistic terms and its players are largely unknown. The general transcription factor II H (TFIIH) is a major...
Evolutionary etiology of high-grade astrocytomas [Genetics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Glioblastoma (GBM), the most common brain malignancy, remains fatal with no effective treatment. Analyses of common aberrations in GBM suggest major regulatory pathways associated with disease etiology. However, 90% of GBMs are diagnosed at an advanced stage (primary GBMs), providing no access to early disease stages for assessing disease progression...
Coordination of broad enhancers by EGFR signaling [Genetics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Although it is widely appreciated that a typical developmental control gene is regulated by multiple enhancers, coordination of enhancer activities remains poorly understood. We propose a mechanism for such coordination in Drosophila oogenesis, when the expression of the transcription factor Broad (BR) evolves from a uniform to a two-domain pattern...
Antitumor immunity induced by CCR4+Treg depletion [Immunology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
CD4+ Treg cells expressing the transcription factor FOXP3 (forkhead box P3) are abundant in tumor tissues and appear to hinder the induction of effective antitumor immunity. A substantial number of T cells, including Treg cells, in tumor tissues and peripheral blood express C-C chemokine receptor 4 (CCR4). Here we show...
Nuclear translation of antigenic peptides [Immunology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The scanning of maturing mRNAs by ribosomes plays a key role in the mRNA quality control process. When ribosomes first engage with the newly synthesized mRNA, and if peptides are produced, is unclear, however. Here we show that ribosomal scanning of prespliced mRNAs occurs in the nuclear compartment, and that...
ILT4 exocytosis regulates neutrophil functions [Immunology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during...
Inflammasome activation by RNA viruses [Immunology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Nod-like receptor family, pyrin domain-containing 3 (NLRP3), is involved in the early stages of the inflammatory response by sensing cellular damage or distress due to viral or bacterial infection. Activation of NLRP3 triggers its assembly into a multimolecular protein complex, termed “NLRP3 inflammasome.” This event leads to the activation of...
ASPP2 suppresses SCC by repressing p63 via NF-{kappa}B [Medical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Squamous cell carcinoma (SCC) is highly malignant and refractory to therapy. The majority of existing mouse SCC models involve multiple gene mutations. Very few mouse models of spontaneous SCC have been generated by a single gene deletion. Here we report a haploinsufficient SCC mouse model in which exon 3 of...
Role of ERR{alpha} in DEN-induced HCC [Medical Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Estrogen-related receptor α (ERRα) is a key regulator of mitochondrial function and metabolism essential for energy-driven cellular processes in both normal and cancer cells. ERRα has also been shown to mediate bone-derived macrophage activation by proinflammatory cytokines. However, the role of ERRα in cancer in which inflammation acts as a...
Small molecule inhibition of quorum sensing [Microbiology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Quorum sensing is a chemical communication process that bacteria use to regulate collective behaviors. Disabling quorum-sensing circuits with small molecules has been proposed as a potential strategy to prevent bacterial pathogenicity. The human pathogen Pseudomonas aeruginosa uses quorum sensing to control virulence and biofilm formation. Here, we analyze synthetic molecules...
DNA-uptake complex of V. cholerae [Microbiology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Natural competence for transformation is a mode of horizontal gene transfer that is commonly used by bacteria to take up DNA from their environment. As part of this developmental program, so-called competence genes, which encode the components of a DNA-uptake machinery, are expressed. Several models have been proposed for the...
Neural correlates of unilateral vestibular lesion [Neuroscience] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Signals from the bilateral vestibular labyrinths work in tandem to generate robust estimates of our motion and orientation in the world. The relative contributions of each labyrinth to behavior, as well as how the brain recovers after unilateral peripheral damage, have been characterized for motor reflexes, but never for perceptual...
Dopamine and NMDA receptor surface interplay [Neuroscience] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of...
On the origin of high-Tc superconductivity [Physics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Unconventional superconductivity (SC) is said to occur when Cooper pair formation is dominated by repulsive electron–electron interactions, so that the symmetry of the pair wave function is other than an isotropic s-wave. The strong, on-site, repulsive electron–electron interactions that are the proximate cause of such SC are more typically drivers...
Universal features in the ARPES of HTSCs [Physics] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
The energy gap for electronic excitations is one of the most important characteristics of the superconducting state, as it directly reflects the pairing of electrons. In the copper–oxide high-temperature superconductors (HTSCs), a strongly anisotropic energy gap, which vanishes along high-symmetry directions, is a clear manifestation of the d-wave symmetry of...
Slowed pacemaking in aged sinoatrial myocytes [Physiology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
An inexorable decline in maximum heart rate (mHR) progressively limits human aerobic capacity with advancing age. This decrease in mHR results from an age-dependent reduction in “intrinsic heart rate” (iHR), which is measured during autonomic blockade. The reduced iHR indicates, by definition, that pacemaker function of the sinoatrial node is...
PHL regulates flowering by bridging phyB and CO [Plant Biology] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
In flowering plants, light is one of the major environmental stimuli that determine the timing of the transition from the vegetative to reproductive phase. In Arabidopsis, phytochrome B (phyB); phyA; cryptochrome 2; and FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 are major photoreceptors that regulate flowering. Unlike phyA; cryptochrome 2; and FLAVIN-BINDING,...
Making music reduces perceived exertion [Psychological and Cognitive Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Music is known to be capable of reducing perceived exertion during strenuous physical activity. The current interpretation of this modulating effect of music is that music may be perceived as a diversion from unpleasant proprioceptive sensations that go along with exhaustion. Here we investigated the effects of music on perceived...
Placebo improvement of pleasant and painful touch [Psychological and Cognitive Sciences] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
Placebo analgesia is often conceptualized as a reward mechanism. However, by targeting only negative experiences, such as pain, placebo research may tell only half the story. We compared placebo improvement of painful touch (analgesia) with placebo improvement of pleasant touch (hyperhedonia) using functional MRI and a crossover design. Somatosensory processing...
Correction for Marcus, Theory of mass-independent fractionation of isotopes, phase space accessibility, and a role of isotopic symmetry [Correction] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
PERSPECTIVE Correction for “Theory of mass-independent fractionation of isotopes, phase space accessibility, and a role of isotopic symmetry,” by Rudolph A. Marcus, which appeared in issue 44, October 29, 2013, of Proc Natl Acad Sci USA (110:17703–17707; first published June 28, 2013; 10.1073/pnas.1213080110). The author notes that, on page 1,...
Correction for Allen et al., Measurements of methane emissions at natural gas production sites in the United States [Correction] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
ENVIRONMENTAL SCIENCES Correction for “Measurements of methane emissions at natural gas production sites in the United States,” by David T. Allen, Vincent M. Torres, James Thomas, David W. Sullivan, Matthew Harrison, Al Hendler, Scott C. Herndon, Charles E. Kolb, Matthew P. Fraser, A. Daniel Hill, Brian K. Lamb, Jennifer Miskimins,...
Correction for Singh et al., mitoBKCa is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location [Correction] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
PHYSIOLOGY Correction for “mitoBKCa is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location,” by Harpreet Singh, Rong Lu, Jean C. Bopassa, Andrea L. Meredith, Enrico Stefani, and Ligia Toro, which appeared in issue 26, June 25, 2013, of Proc Natl Acad Sci USA (110:10836–10841; first...
Mathematics in metal [Science and Culture] [Proceedings of the National Academy of Sciences current issue] 2013-10-29 13:58
A mathematical surface known as the Klein bottle is like a mischievous, mathematical cousin of the Möbius Strip, where the inside and the outside are the same side. Among topologists, the Klein bottle is well known as an example of a closed, nonorientable surface. Thanks to artist Bathsheba Grossman, whose...
This Week in Science [Science: Current Issue] 2013-10-31 20:00
Designer Vaccine | Magnetic Crab | Deep Heating | Quantum Heating | Not All Neurons Are Alike | Better Contact Along the Edge | Prairie Redux | First Defense | The Good Scar | Wiring the Retina | Dinner Time! | Information Physics | Imaging Hydrogen Bonds | Chilly Repression Stalls Flowering | Coherently Controlling Large Cats
[Editorial] Seize the Neuroscience Moment [Science: Current Issue] 2013-10-31 20:00
There seems to be an abundance of neuroscience initiatives right now. This year, the European Commission launched a Human Brain Project, and the U.S. government announced its Brain Research through Advancing Innovative Neurotechnologies (BRAIN) project. They join other recent neuroscience efforts across the world aimed at advancing our understanding of the brain. Exploiting these diverse initiatives to yield scientific, clinical, and economic benefits, however, will require not only political and policy-maker support but also endorsement and extensive involvement by the neuroscience community, which already saw a “Decade of the Brain” come and go about 20 years ago, with little direct result. What's different now? Author: Alan I. Leshner
Editors' Choice [Science: Current Issue] 2013-10-31 20:00
Caribbean Coral | From Drowning to Dried Up | Double Dealing | Better Together | Selective Delivery | Periodic Ions | Bacterial Pockets
[News of the Week] This Week's Section [Science: Current Issue] 2013-10-31 20:00
Follow the links below for a roundup of the week's top stories in science, or download a PDF of the entire section. Around the WorldFindingsRandom SamplesNewsmakers
[News of the Week] Around the World [Science: Current Issue] 2013-10-31 20:00
In science news around the world, inspectors find that Syria's military holds about 1000 tons of chemical precursors to weapons, a Mexican university lifts sanctions against two microbiologists investigated for scientific misconduct, and the European Food Safety Authority again faces conflict of interest questions.
[News of the Week] Random Sample [Science: Current Issue] 2013-10-31 20:00
Roman-era lead salvaged from old shipwrecks is in demand for shielding particle physics experiments—but now, underwater archaeologists worry about creating a market for that lead.
[News of the Week] Newsmakers [Science: Current Issue] 2013-10-31 20:00
Penrose Albright, head of the Lawrence Livermore National Laboratory, announced he'd step down at the end of October.
Findings [Science: Current Issue] 2013-10-31 20:00
A Sense of Snakes | Most Earth-Like Exoplanet Yet
[News & Analysis] New Experiment Torpedoes Lightweight Dark Matter [Science: Current Issue] 2013-10-31 20:00
This week, researchers working with the Large Underground Xenon (LUX) detector at the Sanford Underground Research Facility in Lead, South Dakota, announced that they see no signs of the lightweight dark matter particles hinted at by other experiments. Author: Adrian Cho
[News & Analysis] RNA Helps Resurrect Ancient DNA [Science: Current Issue] 2013-10-31 20:00
A relatively inexpensive way of reading contaminated DNA samples promises to help researchers learn more about the genetics of ancient humans and animals. Author: Jocelyn Kaiser
[News & Analysis] Industry Lobbying Derails Trawling Ban in Europe [Science: Current Issue] 2013-10-31 20:00
Two deep-sea fishing methods cause massive ecological damage, many scientists say. Author: Tania Rabesandratana
[News & Analysis] French Mathematician Tapped to Head Key Funding Agency [Science: Current Issue] 2013-10-31 20:00
French mathematician Jean-Pierre Bourguignon will move to Brussels to become the new president of the European Research Council, Europe's funding agency for basic research. Author: Martin Enserink
[News & Analysis] Structural Biology Triumph Offers Hope Against a Childhood Killer [Science: Current Issue] 2013-10-31 20:00
Fine-grained structural analyses of respiratory syncytial virus have led to a promising new vaccine strategy against this devastating childhood infection. Author: Jon Cohen
[News & Analysis] HIV Surface Proteins Finally Caught Going Au Naturel [Science: Current Issue] 2013-10-31 20:00
Researchers studying HIV have finally engineered a "near native" version of its surface proteins, which promises to clarify the infection process and how to derail it. Author: Jon Cohen
[News Focus] Short-Circuiting Depression [Science: Current Issue] 2013-10-31 20:00
Experimental deep brain stimulation surgeries for depression are giving old theories about the disorder a jolt. Author: Emily Underwood
[News Focus] Dark Matter's Dark Horse [Science: Current Issue] 2013-10-31 20:00
A rare yes/no effort promises to prove either that hypothetical particles called axions are the universe's elusive dark matter—or that they can't be. Author: Adrian Cho
[Letter] Health and Obesity: A New Normal? [Science: Current Issue] 2013-10-31 20:00
Author: Marshall E. Deutsch
[Letter] Health and Obesity: Not Just Skin Deep [Science: Current Issue] 2013-10-31 20:00
Authors: Erik Arner, Peter Arner
[Letter] Emerging Arsenic Threat in Canada [Science: Current Issue] 2013-10-31 20:00
Authors: Victor D. Martinez, Emily A. Vucic, Stephen Lam, Wan L. Lam
[Correction] Corrections and Clarifications [Science: Current Issue] 2013-10-31 20:00
[Letter] Zombiology [Science: Current Issue] 2013-10-31 20:00
Authors: Matthieu J. Guitton, Cécile Cristofari
[Book Review] Deep Inside Champions, Just Genes? [Science: Current Issue] 2013-10-31 20:00
Through a collection of examples, Epstein provides a balanced and nuanced consideration of genetic influences on athletic performance. Authors: Dov Greenbaum, Jieming Chen, Mark Gerstein
[Book Review] An Ethnographic Check-Up [Science: Current Issue] 2013-10-31 20:00
This "ethnographic critique of the contemporary global health enterprise" comprises contributions from anthropologists, historians, an epidemiologist, and a human rights scholar. Author: Nicole S. Berry
[Books et al.] Books Received [Science: Current Issue] 2013-10-31 20:00
A listing of books received at Science during the week ending 25 October 2013.
[Policy Forum] Doctoral Students and U.S. Immigration Policy [Science: Current Issue] 2013-10-31 20:00
Policies should promote skilled non-U.S. students to study, and stay, in the United States. Authors: Keith E. Maskus, A. Mushfiq Mobarak, Eric T. Stuen
[Perspective] Our Fallen Genomes [Science: Current Issue] 2013-10-31 20:00
A human brain can cope with many genomic variations scattered among its neurons. [Also see Report by McConnell et al.] Authors: Evan Z. Macosko, Steven A. McCarroll
[Perspective] Dust Unto Dust [Science: Current Issue] 2013-10-31 20:00
Modern agriculture diminishes the diversity of soil biota, thereby reducing long-term soil fertility. [Also see Report by Fierer et al.] Authors: Mary C. Scholes, Robert J. Scholes
[Perspective] A Pathway to Flowering—Why Staying Cool Matters [Science: Current Issue] 2013-10-31 20:00
Plants time flowering through temperature-sensing mechanisms involving differential RNA abundance, alternative splicing, and protein stability. [Also see Report by Lee et al.] Author: Ove Nilsson
[Perspective] Storing Quantum Information in Schrödinger's Cats [Science: Current Issue] 2013-10-31 20:00
Superposition states created with more than 100 photons enable the storage of multiple bits of quantum information. [Also see Report by Vlastakis et al.] Author: Peter J. Leek
[Perspective] Quantized Electronic Heat Flow [Science: Current Issue] 2013-10-31 20:00
A measurement of the quantum-limited heat flow in an electronic conductor opens a pathway to the nanoscale control of heat currents. [Also see Report by Jezouin et al.] Authors: Björn Sothmann, Christian Flindt
[Perspective] Rhythmic Respiration [Science: Current Issue] 2013-10-31 20:00
Cellular respiration is under circadian control. [Also see Research Article by Peek et al.] Authors: Guillaume Rey, Akhilesh B. Reddy
[Retrospective] David H. Hubel (1926–2013) [Science: Current Issue] 2013-10-31 20:00
A neuroscientist and Nobel laureate transformed our understanding of how input to the brain produces vision and provided new insights into brain development. Author: Robert H. Wurtz
[Essay] Space Bats: Multidimensional Spatial Representation in the Bat [Science: Current Issue] 2013-10-31 20:00
A novel animal model, the bat, is used to elucidate place-cell and grid-cell phenomena. Author: Michael M. Yartsev
[Essay] Play It Again, Brain [Science: Current Issue] 2013-10-31 20:00
Cues associated with a previous experience, presented again during sleep, may improve your memory of this experience. Author: Daniel Bendor
[Essay] Brains Don't Play Dice—or Do They? [Science: Current Issue] 2013-10-31 20:00
Associative brain centers are able to account for an infinite amount of possible associations through randomized sensory input. Author: Sophie J. C. Caron
[Introduction to Special Issue] Connection, Connection, Connection… [Science: Current Issue] 2013-10-31 20:00
Author: Peter Stern
[Special Issue Review] Cortical High-Density Counterstream Architectures [Science: Current Issue] 2013-10-31 20:00
Small-world networks provide an appealing description of cortical architecture owing to their capacity for integration and segregation combined with an economy of connectivity. Previous reports of low-density interareal graphs and apparent small-world properties are challenged by data that reveal high-density cortical graphs in which economy of connections is achieved by weight heterogeneity and distance-weight correlations. These properties define a model that predicts many binary and weighted features of the cortical network including a core-periphery, a typical feature of self-organizing information processing systems. Feedback and feedforward pathways between areas exhibit a dual counterstream organization, and their integration into local circuits constrains cortical computation. Here, we propose a bow-tie representation of interareal architecture derived from the hierarchical laminar weights of pathways between the high-efficiency dense core and periphery. Authors: Nikola T. Markov, Mária Ercsey-Ravasz, David C. Van Essen, Kenneth Knoblauch, Zoltán Toroczkai, Henry Kennedy
[Special Issue Review] Structural and Functional Brain Networks: From Connections to Cognition [Science: Current Issue] 2013-10-31 20:00
How rich functionality emerges from the invariant structural architecture of the brain remains a major mystery in neuroscience. Recent applications of network theory and theoretical neuroscience to large-scale brain networks have started to dissolve this mystery. Network analyses suggest that hierarchical modular brain networks are particularly suited to facilitate local (segregated) neuronal operations and the global integration of segregated functions. Although functional networks are constrained by structural connections, context-sensitive integration during cognition tasks necessarily entails a divergence between structural and functional networks. This degenerate (many-to-one) function-structure mapping is crucial for understanding the nature of brain networks. The emergence of dynamic functional networks from static structural connections calls for a formal (computational) approach to neuronal information processing that may resolve this dialectic between structure and function. Authors: Hae-Jeong Park, Karl Friston
[Special Issue Review] Functional Interactions as Big Data in the Human Brain [Science: Current Issue] 2013-10-31 20:00
Noninvasive studies of human brain function hold great potential to unlock mysteries of the human mind. The complexity of data generated by such studies, however, has prompted various simplifying assumptions during analysis. Although this has enabled considerable progress, our current understanding is partly contingent upon these assumptions. An emerging approach embraces the complexity, accounting for the fact that neural representations are widely distributed, neural processes involve interactions between regions, interactions vary by cognitive state, and the space of interactions is massive. Because what you see depends on how you look, such unbiased approaches provide the greatest flexibility for discovery. Author: Nicholas B. Turk-Browne
[Special Issue Review] Predispositions and Plasticity in Music and Speech Learning: Neural Correlates and Implications [Science: Current Issue] 2013-10-31 20:00
Speech and music are remarkable aspects of human cognition and sensory-motor processing. Cognitive neuroscience has focused on them to understand how brain function and structure are modified by learning. Recent evidence indicates that individual differences in anatomical and functional properties of the neural architecture also affect learning and performance in these domains. Here, neuroimaging findings are reviewed that reiterate evidence of experience-dependent brain plasticity, but also point to the predictive validity of such data in relation to new learning in speech and music domains. Indices of neural sensitivity to certain stimulus features have been shown to predict individual rates of learning; individual network properties of brain activity are especially relevant in this regard, as they may reflect anatomical connectivity. Similarly, numerous studies have shown that anatomical features of auditory cortex and other structures, and their anatomical connectivity, are predictive of new sensory-motor learning ability. Implications of this growing body of literature are discussed. Author: Robert J. Zatorre
[Research Article] On and Off Retinal Circuit Assembly by Divergent Molecular Mechanisms [Science: Current Issue] 2013-10-31 20:00
Work in mice reveals how motion-detection circuitry is established during visual system development. Authors: Lu O. Sun, Zheng Jiang, Michal Rivlin-Etzion, Randal Hand, Colleen M. Brady, Ryota L. Matsuoka, King-Wai Yau, Marla B. Feller, Alex L. Kolodkin
[Research Article] Circadian Clock NAD+ Cycle Drives Mitochondrial Oxidative Metabolism in Mice [Science: Current Issue] 2013-10-31 20:00
The coenzyme nicotinamide adenine dinucleotide mechanistically links the circadian clock to control of energy production by mitochondria. [Also see Perspective by Rey and Reddy] Authors: Clara Bien Peek, Alison H. Affinati, Kathryn Moynihan Ramsey, Hsin-Yu Kuo, Wei Yu, Laura A. Sena, Olga Ilkayeva, Biliana Marcheva, Yumiko Kobayashi, Chiaki Omura, Daniel C. Levine, David J. Bacsik, David Gius, Christopher B. Newgard, Eric Goetzman, Navdeep S. Chandel, John M. Denu, Milan Mrksich, Joseph Bass
[Research Article] Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus [Science: Current Issue] 2013-10-31 20:00
Molecular engineering of a childhood virus surface protein significantly improves protective responses in mice and macaques. Authors: Jason S. McLellan, Man Chen, M. Gordon Joyce, Mallika Sastry, Guillaume B. E. Stewart-Jones, Yongping Yang, Baoshan Zhang, Lei Chen, Sanjay Srivatsan, Anqi Zheng, Tongqing Zhou, Kevin W. Graepel, Azad Kumar, Syed Moin, Jeffrey C. Boyington, Gwo-Yu Chuang, Cinque Soto, Ulrich Baxa, Arjen Q. Bakker, Hergen Spits, Tim Beaumont, Zizheng Zheng, Ningshao Xia, Sung-Youl Ko, John-Paul Todd, Srinivas Rao, Barney S. Graham, Peter D. Kwong
[Report] Evolution of the Magnetic Field Structure of the Crab Pulsar [Science: Current Issue] 2013-10-31 20:00
Long-term measurements show the systematic evolution of the radiation pattern of one of the youngest neutron stars known. Authors: Andrew Lyne, Francis Graham-Smith, Patrick Weltevrede, Christine Jordan, Ben Stappers, Cees Bassa, Michael Kramer
[Report] Quantum Limit of Heat Flow Across a Single Electronic Channel [Science: Current Issue] 2013-10-31 20:00
The unit of heat carried by electrons is measured using noise thermometry and found to be consistent with predictions. [Also see Perspective by Sothmann and Flindt] Authors: S. Jezouin, F. D. Parmentier, A. Anthore, U. Gennser, A. Cavanna, Y. Jin, F. Pierre
[Report] Parameter Space Compression Underlies Emergent Theories and Predictive Models [Science: Current Issue] 2013-10-31 20:00
An information-theoretical approach is used to distinguish the important parameters in two archetypical physics models. Authors: Benjamin B. Machta, Ricky Chachra, Mark K. Transtrum, James P. Sethna
[Report] Deterministically Encoding Quantum Information Using 100-Photon Schrödinger Cat States [Science: Current Issue] 2013-10-31 20:00
A scheme is demonstrated for coherently mapping the state of a single superconducting qubit onto a large number of photons. [Also see Perspective by Leek] Authors: Brian Vlastakis, Gerhard Kirchmair, Zaki Leghtas, Simon E. Nigg, Luigi Frunzio, S. M. Girvin, Mazyar Mirrahimi, M. H. Devoret, R. J. Schoelkopf
[Report] Real-Space Identification of Intermolecular Bonding with Atomic Force Microscopy [Science: Current Issue] 2013-10-31 20:00
An atomic force microscope tip bearing a single carbon monoxide molecule was used to resolve hydrogen-bonding contacts between molecules. Authors: Jun Zhang, Pengcheng Chen, Bingkai Yuan, Wei Ji, Zhihai Cheng, Xiaohui Qiu
[Report] One-Dimensional Electrical Contact to a Two-Dimensional Material [Science: Current Issue] 2013-10-31 20:00
Metal contacts to graphene along its edge improve bonding and, in turn, electronic performance. Authors: L. Wang, I. Meric, P. Y. Huang, Q. Gao, Y. Gao, H. Tran, T. Taniguchi, K. Watanabe, L. M. Campos, D. A. Muller, J. Guo, P. Kim, J. Hone, K. L. Shepard, C. R. Dean
[Report] Pacific Ocean Heat Content During the Past 10,000 Years [Science: Current Issue] 2013-10-31 20:00
Marine records show how ocean heat content has varied in step with climate over the past 10,000 years. Authors: Yair Rosenthal, Braddock K. Linsley, Delia W. Oppo
[Report] Reconstructing the Microbial Diversity and Function of Pre-Agricultural Tallgrass Prairie Soils in the United States [Science: Current Issue] 2013-10-31 20:00
Analysis of microbiota in prairie soil relicts offers insights into the ecological function of a near-extinct biome. [Also see Perspective by Scholes and Scholes] Authors: Noah Fierer, Joshua Ladau, Jose C. Clemente, Jonathan W. Leff, Sarah M. Owens, Katherine S. Pollard, Rob Knight, Jack A. Gilbert, Rebecca L. McCulley
[Report] Structural Basis for flg22-Induced Activation of the Arabidopsis FLS2-BAK1 Immune Complex [Science: Current Issue] 2013-10-31 20:00
The molecular basis for how a plant heterodimeric receptor responds to bacterial infection signals is elucidated. Authors: Yadong Sun, Lei Li, Alberto P. Macho, Zhifu Han, Zehan Hu, Cyril Zipfel, Jian-Min Zhou, Jijie Chai
[Report] Regulation of Temperature-Responsive Flowering by MADS-Box Transcription Factor Repressors [Science: Current Issue] 2013-10-31 20:00
A warm spring favors early flowering by invoking less transcriptional repression by a floral repressor complex. [Also see Perspective by Nilsson] Authors: Jeong Hwan Lee, Hak-Seung Ryu, Kyung Sook Chung, David Posé, Soonkap Kim, Markus Schmid, Ji Hoon Ahn
[Report] Mosaic Copy Number Variation in Human Neurons [Science: Current Issue] 2013-10-31 20:00
Single-cell genomics reveals that individual adult human neurons acquire diverse individual genomes. [Also see Perspective by Macosko and McCarroll] Authors: Michael J. McConnell, Michael R. Lindberg, Kristen J. Brennand, Julia C. Piper, Thierry Voet, Chris Cowing-Zitron, Svetlana Shumilina, Roger S. Lasken, Joris R. Vermeesch, Ira M. Hall, Fred H. Gage
[Report] Resident Neural Stem Cells Restrict Tissue Damage and Neuronal Loss After Spinal Cord Injury in Mice [Science: Current Issue] 2013-10-31 20:00
Glial scarring helps to maintain the integrity of the injured spinal cord in mice. Authors: Hanna Sabelström, Moa Stenudd, Pedro Réu, David O. Dias, Marta Elfineh, Sofia Zdunek, Peter Damberg, Christian Göritz, Jonas Frisén
[New Products] New Products [Science: Current Issue] 2013-10-31 20:00
A weekly roundup of information on newly offered instrumentation, apparatus, and laboratory materials of potential interest to researchers.
Construction of microRNA functional families by a mixture model of position weight matrices [PeerJ] 2013-10-30 20:00
MicroRNAs (miRNAs) are small regulatory molecules that repress the translational processes of their target genes by binding to their 3′ untranslated regions (3′ UTRs). Because the target genes are predominantly determined by their sequence complementarity to the miRNA seed regions (nucleotides 2–7) which are evolutionarily conserved, it is inferred that the target relationships and functions of the miRNA family members are conserved across many species. Therefore, detecting the relevant miRNA families with confidence would help to clarify the conserved miRNA functions, and elucidate miRNA-mediated biological processes. We present a mixture model of position weight matrices for constructing miRNA functional families. This model systematically finds not only evolutionarily conserved miRNA family members but also functionally related miRNAs, as it simultaneously generates position weight matrices representing the conserved sequences. Using mammalian miRNA sequences, in our experiments, we identified potential miRNA groups characterized by similar sequence patterns that have common functions. We validated our results using score measures and by the analysis of the conserved targets. Our method would provide a way to comprehensively identify conserved miRNA functions.
Predicting pKa for proteins using COSMO-RS [PeerJ] 2013-10-30 20:00
We have used the COSMO-RS implicit solvation method to calculate the equilibrium constants, pKa, for deprotonation of the acidic residues of the ovomucoid inhibitor protein, OMTKY3. The root mean square error for comparison with experimental data is only 0.5 pH units and the maximum error 0.8 pH units. The results show that the accuracy of pKa prediction using COSMO-RS is as good for large biomolecules as it is for smaller inorganic and organic acids and that the method compares very well to previous pKa predictions of the OMTKY3 protein using Quantum Mechanics/Molecular Mechanics. Our approach works well for systems of about 1000 atoms or less, which makes it useful for small proteins as well as for investigating portions of larger proteins such as active sites in enzymes.
An extended genovo metagenomic assembler by incorporating paired-end information [PeerJ] 2013-10-30 20:00
Metagenomes present assembly challenges, when assembling multiple genomes from mixed reads of multiple species. An assembler for single genomes can’t adapt well when applied in this case. A metagenomic assembler, Genovo, is a de novo assembler for metagenomes under a generative probabilistic model. Genovo assembles all reads without discarding any reads in a preprocessing step, and is therefore able to extract more information from metagenomic data and, in principle, generate better assembly results. Paired end sequencing is currently widely-used yet Genovo was designed for 454 single end reads. In this research, we attempted to extend Genovo by incorporating paired-end information, named Xgenovo, so that it generates higher quality assemblies with paired end reads.
First, we extended Genovo by adding a bonus parameter in the Chinese Restaurant Process used to get prior accounts for the unknown number of genomes in the sample. This bonus parameter intends for a pair of reads to be in the same contig and as an effort to solve chimera contig case. Second, we modified the sampling process of the location of a read in a contig. We used relative distance for the number of trials in the symmetric geometric distribution instead of using distance between the offset and the center of contig used in Genovo. Using this relative distance, a read sampled in the appropriate location has higher probability. Therefore a read will be mapped in the correct location.
Results of extensive experiments on simulated metagenomic datasets from simple to complex with species coverage setting following uniform and lognormal distribution showed that Xgenovo can be superior to the original Genovo and the recently proposed metagenome assembler for 454 reads, MAP. Xgenovo successfully generated longer N50 than Genovo and MAP while maintaining the assembly quality even for very complex metagenomic datasets consisting of 115 species. Xgenovo also demonstrated the potential to decrease the computational cost. This means that our strategy worked well. The software and all simulated datasets are publicly available online at http://xgenovo.dna.bio.keio.ac.jp.
Prolactin and fMRI response to SKF38393 in the baboon [PeerJ] 2013-10-28 20:00
Background. This study’s goal was to provide dose–response data for a dopamine agonist in the baboon using standard methods (replicate measurements at each dose, across a range of doses), as a standard against which to subsequently validate a novel pharmacological MRI (phMRI) method. Dependent variables were functional MRI (fMRI) data from brain regions selected a priori, and systemic prolactin release. Necessary first steps included estimating the magnitude and time course of prolactin response to anesthesia alone and to various doses of agonist. These first steps (“time course studies”) were performed with three agonists, and the results were used to select promising agonists and to guide design details for the single-dose studies needed to generate dose–response curves.
Methods. We studied 6 male baboons (Papio anubis) under low-dose isoflurane anesthesia after i.m. ketamine. Time course studies charted the changes in plasma prolactin levels over time after anesthesia alone or after an intravenous (i.v.) dose of the dopamine D1-like agonists SKF82958 and SKF38393 or the D2-like agonist pramipexole. In the single-dose dopamine agonist studies, one dose of SKF38393 (ranging from 0.0928–9.28 mg/kg, N = 5 animals) or pramipexole (0.00928–0.2 mg/kg, N = 1) was given i.v. during a 40-min blood oxygen level dependent (BOLD) fMRI session, to determine BOLD and plasma prolactin responses to different drug concentrations. BOLD response was quantified as the area under the time-signal curve for the first 15 min after the start of the drug infusion, compared to the linearly predicted signal from the baseline data before drug. The ED50 (estimated dose that produces 50% of the maximal possible response to drug) for SKF38393 was calculated for the serum prolactin response and for phMRI responses in hypothalamus, pituitary, striatum and midbrain.
Results. Prolactin rose 2.4- to 12-fold with anesthesia alone, peaking around 50–90 min after ketamine administration and gradually tapering off but still remaining higher than baseline on isoflurane 3–5 h after ketamine. Baseline prolactin level increased with age. SKF82958 0.1 mg/kg i.v. produced no noticeable change in plasma prolactin concentration. SKF38393 produced a substantial increase in prolactin release that peaked at around 20–30 min and declined to pre-drug levels in about an hour. Pramipexole quickly reduced prolactin levels below baseline, reaching a nadir 2–3 h after infusion. SKF38393 produced clear, dose-responsive BOLD signal changes, and across the four regions, ED50 was estimated at 2.6–8.1 mg/kg.
Conclusions. In the baboon, the dopamine D1 receptor agonist SKF38393 produces clear plasma prolactin and phMRI dose–response curves. Variability in age and a modest sample size limit the precision of the conclusions.
Vertical and horizontal distribution of Desmophyllum dianthus in Comau Fjord, Chile: a cold-water coral thriving at low pH [PeerJ] 2013-10-28 20:00
Cold-water corals provide an important habitat for a rich fauna along the continental margins and slopes. Although these azooxanthellate corals are considered particularly sensitive to ocean acidification, their responses to natural variations in pH and aragonite saturation are largely unknown due to the difficulty of studying their ecology in deep waters. Previous SCUBA investigations have shown an exceptionally shallow population of the cold-water coral Desmophyllum dianthus in near-surface waters of Comau Fjord, a stratified 480 m deep basin in northern Chilean Patagonia with suboxic deep waters. Here, we use a remotely operated vehicle to quantitatively investigate the distribution of D. dianthus and its physico-chemical drivers in so far uncharted naturally acidified waters. Remarkably, D. dianthus was ubiquitous throughout the fjord, but particularly abundant between 20 and 280 m depth in a pH range of 8.4 to 7.4. The persistence of individuals in aragonite-undersaturated waters suggests that present-day D. dianthus in Comau Fjord may show pre-acclimation or pre-adaptation to conditions of ocean acidification predicted to reach over 70% of the known deep-sea coral locations by the end of the century.
Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid [PeerJ] 2013-10-28 20:00
Cell density is the critical parameter controlling tendon morphogenesis. Knowing its neighbors allows a cell to regulate correctly its proliferation and collagen production. A missing link to understanding this process is a molecular description of the sensing mechanism. Previously, this mechanism was shown in cell culture to rely on a diffusible factor (SNZR [sensor]) with an affinity for the cell layer. This led to purifying conditioned medium over 4 columns and analyzing the final column fractions for band intensity on SDS gels versus biological activity – a 16 kD band strongly correlated between assays. N-terminal sequencing – EPLAVVDL – identified a large gene (424 AA), extremely conserved between chicken and human. In this paper we probe whether this is the correct gene. Can the predicted large protein be cleaved to a smaller protein? EPLAVVDL occurs towards the C-terminus and cleavage would create a small 94 AA protein. This protein would run at ∼10 kD, so what modifications or cofactor binding accounts for its running at 16 kD on SDS gels? This protein has no prominent hydrophobic regions, so can it be secreted? To validate its role, the chicken cDNA for this gene was tagged with myc and his and transfected into a human osteosarcoma cell line (U2OS). U2OS cells expressed the gene but not passively: differentiating into structures resembling spongy bone and expressing alkaline phosphatase, an early bone marker. Intracellularly, two bands were observed by Western blotting: the full length protein and a smaller form (26 kD). Outside the cell, a small band (28 kD) was detected, although it was 40% larger than expected, as well as multiple larger bands. These larger forms could be converted to the predicted smaller protein (94 AA + tags) by changing salt concentrations and ultrafiltering – releasing a cofactor to the filtrate while leaving a protein factor in the retentate. Using specific degradative enzymes and mass spectrometry, the bone cofactor was identified as a lipid containing a ceramide phosphate, a single chained glycerol lipid and a linker. Tendon uses a different cofactor made up of two fatty acid chains linked directly to the phosphate yielding a molecule about half the size. Moreover, adding the tendon factor/cofactor to osteosarcoma cells causes them to stop growing, which is opposite to its role with tendon cells. Thus, the cofactor is cell type specific both in composition and in the triggered response. Further support of its proposed role came from frozen sections from 5 week old mice where an antibody to the factor stained strongly at the growing ends of the tendon as predicted. In conclusion, the molecule needed for cell density signaling is a small protein bound to a unique, tissue-specific phospholipid yielding a membrane associated but diffusible molecule. Signal transduction is postulated to occur by an increased ordering of the plasma membrane as the concentration of this protein/lipid increases with cell density.
A new species of the archaic primate Zanycteris from the late Paleocene of western Colorado and the phylogenetic position of the family Picrodontidae [PeerJ] 2013-10-28 20:00
A new species of an archaic primate (Pleisadapiformes) is described based on a maxilla containing the first and second upper molars from the Fort Union Formation, Atwell Gulch Member in northwestern Colorado. The preserved teeth show the unusual dental characteristics of members of the rare and poorly documented Picrodontidae family, including an elongated centrocrista and wide occlusal surface. The new species is placed within the genus Zanycteris (represented by a single specimen from southern Colorado). This placement is based on similarities in regard to the parastyle, curvilinear centrocrista, and wider anterior stylar shelf on the upper molars. However, the new species differs from the only known species of Zanycteris in exhibiting an upper first molar that is 30% larger in area, while retaining a similarly sized upper second molar. Phylogenetic analysis supports the separation of the Picrodontidae family from the Paromomyidae, while still recognizing picrodontids position within Pleisadapiformes. The unusual dental features of the upper molars likely functioned in life as an enhanced shearing surface between the centrocrista and cristid obliqua crests for a specialized diet of fruit. A similar arrangement is found in the living bat Ariteus (Jamaican fig-eating bat), which feeds on fleshy fruit. The new species showcases the rapid diversification of archaic primates shortly after the extinction of the dinosaurs during the Paleocene, and the unusual dental anatomy of picrodontids to exploit new dietary specializations.
BioNames: linking taxonomy, texts, and trees [PeerJ] 2013-10-28 20:00
BioNames is a web database of taxonomic names for animals, linked to the primary literature and, wherever possible, to phylogenetic trees. It aims to provide a taxonomic “dashboard” where at a glance we can see a summary of the taxonomic and phylogenetic information we have for a given taxon and hence provide a quick answer to the basic question “what is this taxon?” BioNames combines classifications from the Global Biodiversity Information Facility (GBIF) and GenBank, images from the Encyclopedia of Life (EOL), animal names from the Index of Organism Names (ION), and bibliographic data from multiple sources including the Biodiversity Heritage Library (BHL) and CrossRef. The user interface includes display of full text articles, interactive timelines of taxonomic publications, and zoomable phylogenies. It is available at http://bionames.org.
Using multi-scale distribution and movement effects along a montane highway to identify optimal crossing locations for a large-bodied mammal community [PeerJ] 2013-10-23 20:00
Roads are a major cause of habitat fragmentation that can negatively affect many mammal populations. Mitigation measures such as crossing structures are a proposed method to reduce the negative effects of roads on wildlife, but the best methods for determining where such structures should be implemented, and how their effects might differ between species in mammal communities is largely unknown. We investigated the effects of a major highway through south-eastern British Columbia, Canada on several mammal species to determine how the highway may act as a barrier to animal movement, and how species may differ in their crossing-area preferences. We collected track data of eight mammal species across two winters, along both the highway and pre-marked transects, and used a multi-scale modeling approach to determine the scale at which habitat characteristics best predicted preferred crossing sites for each species. We found evidence for a severe barrier effect on all investigated species. Freely-available remotely-sensed habitat landscape data were better than more costly, manually-digitized microhabitat maps in supporting models that identified preferred crossing sites; however, models using both types of data were better yet. Further, in 6 of 8 cases models which incorporated multiple spatial scales were better at predicting preferred crossing sites than models utilizing any single scale. While each species differed in terms of the landscape variables associated with preferred/avoided crossing sites, we used a multi-model inference approach to identify locations along the highway where crossing structures may benefit all of the species considered. By specifically incorporating both highway and off-highway data and predictions we were able to show that landscape context plays an important role for maximizing mitigation measurement efficiency. Our results further highlight the need for mitigation measures along major highways to improve connectivity between mammal populations, and illustrate how multi-scale data can be used to identify preferred crossing sites for different species within a mammal community.
Transformative optimisation of agricultural land use to meet future food demands [PeerJ] 2013-10-23 20:00
The human population is expected to reach ∼9 billion by 2050. The ensuing demands for water, food and energy would intensify land-use conflicts and exacerbate environmental impacts. Therefore we urgently need to reconcile our growing consumptive needs with environmental protection. Here, we explore the potential of a land-use optimisation strategy to increase global agricultural production on two major groups of crops: cereals and oilseeds. We implemented a spatially-explicit computer simulation model across 173 countries based on the following algorithm: on any cropland, always produce the most productive crop given all other crops currently being produced locally and the site-specific biophysical, economic and technological constraints to production. Globally, this strategy resulted in net increases in annual production of cereal and oilseed crops from 1.9 billion to 2.9 billion tons (46%), and from 427 million to 481 million tons (13%), respectively, without any change in total land area harvested for cereals or oilseeds. This thought experiment demonstrates that, in theory, more optimal use of existing farmlands could help meet future crop demands. In practice there might be cultural, social and institutional barriers that limit the full realisation of this theoretical potential. Nevertheless, these constraints have to be weighed against the consequences of not producing enough food, particularly in regions already facing food shortages.
Free Flow of Sweat due to Loss of Surface Tension at Sweat Droplet Causes Water-Induced Skin Wrinkling [PeerJ Preprints] 2013-10-29 20:00
Water immersion skin wrinkling appears to be the result of breaking the balance between secretory pressure of sweat glands and the pressure exerted by the surface tension of sweat droplet at the pore. When a hand is immersed in water, sweat droplet easily merge within the water causing pressure to drop at the pore. The resulted imbalance in pressure enables the sweat to flow freely into the water. Flow of sweat continues as long as there is a blood flow to hand. To prevent the loss of sweat from the body and to maintain homeostasis, sympathetic nerves trigger the reduction of blood flow to hand causing vasoconstriction. The overlying skin wrinkles due to loss of volume under the skin.
A within-subject comparison of seizure duration with etomidate and methohexital in electroconvulsive therapy [PeerJ Preprints] 2013-10-29 20:00
Objective: To determine whether etomidate is associated with longer seizures than methohexital in ECT anesthesia. Methods: Retrospective chart review in 39 patients who were switched from one anesthetic to the other. We compared motor and EEG seizure duration in the last ECT session on one anesthetic and the first session on the other anesthetic. Results: Motor seizures were about 10 seconds longer with etomidate (p < 0.05). However, few of the increases in seizure duration had obvious clinical import. Conclusions: Etomidate can lengthen ECT seizure time compared with methohexital, but the clinical significance of this observation requires further study.
Association between shell morphology of micro-land snails (genus Plectostoma) and their predator’s predatory behaviour [PeerJ Preprints] 2013-10-23 20:00
Predator-prey interactions are among the main ecological interactions that shape the diversity of biological form. In many cases, the evolution of the mollusc shell form is presumably driven by predation. However, the adaptive significance of several uncommon, yet striking, shell traits of land snails are still poorly known. These include the distorted coiled “tuba” and the protruded radial ribs that can be found in micro-landsnails of the genus Plectostoma. Here, we experimentally tested whether these shell traits may act as defensive adaptations against predators. First, we identified the predators, namely, Atopos slugs and Pteroptyx beetle larvae, and their predatory strategies towards Plectostoma snails. Then, we characterised and quantified the possible anti-predation behaviour and shell traits of Plectostoma snails both in terms of their properties and efficiencies in defending against the Atopos slug predatory strategies, namely, shell-apertural entry and shell-drilling. The results showed that Atopos slugs would first attack the snail by shell-apertural entry, and, should this fail, shift to the energetically more costly shell-drilling strategy. We found that the shell tuba of Plectostoma snails is an effective defensive trait against shell-apertural entry attack. None of the snail traits, such as resting behaviour, shell thickness, shell tuba shape, shell rib density and intensity can protect the snail from the slug’s shell-drilling attack. However, these traits could increase the predation costs to the slug. Further analysis on the shell traits revealed that the lack of effectiveness these anti-predation shell traits may be caused by a functional trade-off between shell traits under selection of two different predatory strategies. Lastly, we discuss our results in the framework of Red Queen predator-prey coevolution and escalation, and propose several key elements for future study.
Whale Shark Aggregations in the Northern Gulf of Mexico [PeerJ Preprints] 2013-10-21 20:00
Background: The Northern Gulf of Mexico Whale Shark Research Program was initiated in 2003 to increase our knowledge of whale shark occurrence and distribution within the region. A primary goal was to obtain sightings data from “citizen scientists” to guide directed research. Whale sharks are typically solitary animals, but are known to aggregate in areas of high prey abundance. Methods: Whale shark sightings data reported from 2003 to present were compiled. Aggregations were defined as more than one whale shark, and large aggregations were defined as 10 or more sharks, observed at a specific location at the same time. Efforts were made to encounter aggregations to determine size and sex assemblage of the sharks, and to collect plankton samples to identify potential prey. Results: To date we have over 600 whale shark sightings reports and four research encounters of large aggregations. Whale shark aggregations represented 31% of reported sightings, 25% of which were large aggregations between 10-150 individuals. All of thereported large aggregations occurred during summer, almost exclusively along the continental shelf edge, with 41% occurring at Ewing Bank. Three out of the four scientific encounters occurred at Ewing Bank (2009, 2010, 2013). Aggregations were dominated by immature males that were primarily feeding on Euthynnus alletteratus eggs. Conclusions: The use of sightings data provided by “citizen scientists” has proven to bean inexpensive and effective technique for identifying whale shark aggregation locations in the northern Gulf of Mexico. Aggregation assemblages appear to consist of largely juvenile males that were feeding on tuna eggs. Similar to other regions, it is unknown if whale sharks in the northern Gulf of Mexico primarily consist of juveniles or if juveniles simply dominate these large feeding aggregations.
The Bioinformatics Open Source Conference (BOSC) 2013 [PeerJ Preprints] 2013-10-19 20:00
The 14th annual Bioinformatics Open Source Conference (BOSC) was held in Berlin in July 2013, bringing together over 100 bioinformatics researchers, developers and users of open source software. Since its inception in 2000, BOSC has been organised as a Special Interest Group (SIG) satellite meeting preceding the large International Conference on Intelligent Systems for Molecular Biology (ISMB), which is the annual meeting of the International Society for Computational Biology (ISCB). BOSC provides bioinformatics developers with a forum for communicating the results of their latest efforts to the wider research community, and a focused environment for developers and users to interact and share ideas about standards, software development practices, and practical techniques for solving bioinformatics problems. As in previous years, BOSC 2013 was preceded by a Codefest, a two day hackathon that brings together bioinformatics open source project developers and members of the community and allows them to work collaboratively and achieve greater interoperability between tools developed by different groups.
The session topics at BOSC 2013 included several that have been popular in previous years, including Cloud and Parallel Computing, Visualization, Software Interoperability, Genome-scale Data Management, and a session for updates on ongoing open source projects, as well as two new sessions: Translational Bioinformatics, recognizing the growing use of computational biology in medical applications, and Open Science and Reproducible Research. Open Science, a movement dedicated to making all aspects of scientific knowledge production freely available for reuse and extension, not only validates published results by allowing others to reproduce them, but also accelerates the pace of scientific discovery by enabling researchers to more efficiently build on previous work, rather than having to reinvent tools and reassemble data sets.
BOSC typically features two keynote talks by researchers who are influential in some aspect of open source bioinformatics. Our first keynote talk this year was by Cameron Neylon, the Advocacy Director for the Public Library of Science (PLOS), who is a prominent advocate for open science. He discussed the cultural issues that are hindering open science, and how openness in scientific collaborations can generate impact. Our second keynote speaker, Sean Eddy, who is perhaps best known as the author of the HMMER software suite, began his keynote talk with an inspiring history of how he got involved in bioinformatics and proceeded to argue that dedicating effort to thorough engineering in tool development, which is often shunned as incremental, can become the key to creating a lasting impact.
With the increasing reliance of more and more fields of biology on computational tools to manage and analyze their data, BOSC is well positioned to stay relevant to life science, and thus life scientists, for many years to come.
Fitting occupancy models with E-SURGE: Hidden Markov modelling of presence-absence data [PeerJ Preprints] 2013-10-19 20:00
1. Occupancy – the proportion of area occupied by a species – is a key notion for addressing important questions in ecology, biogeography and conservation biology. Occupancy models allow estimating and inferring about species occurrence while accounting for false absences (or imperfect species detection). 2. Most occupancy models can be formulated as hidden Markov models (HMM) in which the state process captures the Markovian dynamic of the actual but latent states while the observation process consists of observations that are made from these underlying states. 3. We show how occupancy models can be implemented in program E-SURGE, which was initially developed to analyse capture-recapture data in the HMM framework. Replacing individuals by sites provides the user with access to several features of E-SURGE that are not available altogether or just not available in standard occupancy software: i) user-friendly model specification through a SAS/R-like syntax without having to write custom code, ii) decomposition of the observation and state processes in several steps to provide flexible parameterisation, iii) up-to-date diagnostics of model identifiability and iv) advanced numerical algorithms to produce fast and reliable results (including site random effects). 4. To illustrate E-SURGE features, we provide simulated data and the details of the implementation on the analysis of several occupancy models. These detailed examples are gathered in a companion wiki platform http://occupancyinesurge.wikidot.com/ .
Demographics and feeding ecology of whale sharks at Mafia Island, Tanzania [PeerJ Preprints] 2013-10-16 20:00
Background. The Western Indian Ocean is a globally important region for the whale shark Rhincodon typus, with well-studied coastal aggregation sites in southern Mozambique, Seychelles and Djibouti. Here we present an overview of a new study at Mafia Island, Tanzania. Methods. We monitored whale shark abundances on 103 boat trips from October 2012–March 2013. We also used passive acoustic telemetry (VEMCO® V16 tags) and photographic identification to monitor the residency times and local movements of 29 tagged individuals. Shark sizes were estimated using laser photogrammetry. Results. In total we observed 87 individual sharks with a mean of 5.1 ± 5.2 (± SD) per trip and a peak in December of 8.2 ± 6.3. Total length ranged from 4.1 to 9.7 m and almost all sharks were immature. After boat-based visual observations dropped to zero in March 2013 (with the same ongoing sampling effort), the acoustic array still detected 75% of tagged sharks. Tagged individuals were detected by the acoustic array for 73 ± 40 days on average. They showed a strong site fidelity to a 15 km2 area in the inner part of the bay and then progressively moved offshore at the end of the season, matching a decrease in plankton abundance. Sharks were mostly observed feeding on dense patches of the pelagic shrimp Lucifer hanseni, often in association with planktivorous fishes. Photo IDs from 2007-09 and 2012-13 indicate that a large proportion of the juvenile individuals return to Mafia Island each spring-summer. Conclusion. The size range and gender distribution of whale sharks at Mafia Island is similar to other coastal aggregations in the Indian Ocean, but the relatively high site fidelity and residency time stands in contrast.
The geographic scaling of biotic interactions [PeerJ Preprints] 2013-10-16 20:00
A central tenet of ecology and biogeography is that the broad outlines of species ranges are determined by climate, whereas the effects of biotic interactions are manifested at local scales. While the first proposition is supported by ample evidence, the second is still a matter of controversy. To address this question, we develop a mathematical model that predicts the spatial overlap, i.e., co-occurrence, between pairs of species subject to all possible types of interactions. We then identify the scale in which predicted range overlaps are lost. We found that co-occurrence arising from positive interactions, such as mutualism (+/+) and commensalism (+/0), are manifested across scales of resolution. Negative interactions, such as competition (-/-) and amensalism (-/0), generate checkerboard-type co-occurrence patterns that are discernible at finer resolutions. Scale dependence in consumer-resource interactions (+/-) depends on the strength of positive dependencies between species. Our results challenge the widely held view that climate alone is sufficient to characterize species distributions at broad scales, but also demonstrate that the spatial signature of competition is unlikely to be discernible beyond local and regional scales.
spliceR: An R package for classification of alternative splicing and prediction of coding potential from RNA-seq data. [PeerJ Preprints] 2013-10-10 20:00
With the advent of increasing depth and decreasing costs in digital gene expression technologies exemplified by RNA-sequencing, researchers are now able to profile the transcriptome with unprecedented detail. These advances not only allow for precise approximation of gene expression levels, but also for characterization of alternative isoform usage/switching between samples. Recent software improvements in full transcript deconvolution prompted us to develop spliceR , an R package for classification of alternative splicing. spliceR labels isoforms based on fully assembled transcripts, detecting single- and multiple exon skipping, alternative donor or acceptor sites, intron retention, alternative first or last exon usage, and mutually exclusive exon events. Alongside, event spliced-in/out values are calculated for effective post-filtering, and genomic coordinates of differentially spliced elements are annotated for downstream sequence analysis. Furthermore, spliceR has the option to predict the coding potential and thereby the nonsense mediated decay (NMD) sensitivity of transcripts based on stop codon position.
Bird migratory flyways influence the phylogeography of the invasive brine shrimp Artemia franciscana in its native American range [PeerJ Preprints] 2013-10-08 20:00
Since Darwin’s time, waterbirds have been considered an important vector for the dispersal of continental aquatic invertebrates. Bird movements have facilitated the worldwide invasion of the American brine shrimp Artemia franciscana, transporting cysts (diapausing eggs), and favouring rapid range expansions from introduction sites. Here we address the impact of bird migratory flyways on the population genetic structure and phylogeography of A. franciscana in its native range in the Americas. We examined the sequence variation for two mitochondrial gene fragments (COI and 16S for a subset of the data) in a large set of population samples representing the entire native range of A. franciscana. Furthermore, we performed Mantel tests and redundancy analyses (RDA) to test the role of flyways, geography and human introductions on the phylogeography and population genetic structure at a continental scale. A. franciscanamitochondrial DNA was very diverse, with two main clades, largely corresponding to Pacific and Atlantic populations, mirroring American bird flyways. There was a high degree of regional endemism, with populations subdivided into at least 12 divergent, geographically restricted and largely allopatric mitochondrial lineages, and high levels of population structure ( Φ ST of 0.92), indicating low ongoing gene flow. We found evidence of human-mediated introductions in nine out of 39 populations analysed. Once these populations were removed, Mantel tests revealed a strong association between genetic variation and geographic distance (i.e., isolation-by-distance pattern). RDA showed that shared bird flyways explained around 20% of the variance in genetic distance between populations and this was highly significant, once geographic distance was controlled for. The variance explained increased to 30% when the factor human introduction was included in the model. Our findings suggest that bird-mediated transport of brine shrimp propagules does not result in substantial ongoing gene flow; instead, it had a significant historical role on the current species phylogeography, facilitating the colonisation of new aquatic environments as they become available along their main migratory flyways.
Music Monday: I’ll see you in my dreams [Byte Size Biology] 2013-10-28 07:05
Because.. Django reinhardt.
The Right to Read [Byte Size Biology] 2013-10-24 10:22
Since this is Open Access Week, I thought I’d do the Open-Access / CC thing and share someone else’s work. In this case, a highly topical short story written by Richard Stallman. The author also has a constantly updated page with comments on the restrictions placed today on sharing reading materials. As you will see, this story may be not too far-fetched as it first seems…
The following article appeared in the February 1997 issue of Communications of the ACM (Volume 40, Number 2).
Copyright © 1996, 2002, 2007, 2009, 2010 Richard Stallman
Reproduced under CC-BY-Noderiv license
From The Road To Tycho, a collection of articles about the antecedents of the Lunarian Revolution, published in Luna City in 2096.
For Dan Halbert, the road to Tycho began in college—when Lissa Lenz asked to borrow his computer. Hers had broken down, and unless she could borrow another, she would fail her midterm project. There was no one she dared ask, except Dan.
This put Dan in a dilemma. He had to help her—but if he lent her his computer, she might read his books. Aside from the fact that you could go to prison for many years for letting someone else read your books, the very idea shocked him at first. Like everyone, he had been taught since elementary school that sharing books was nasty and wrong—something that only pirates would do.
And there wasn’t much chance that the SPA—the Software Protection Authority—would fail to catch him. In his software class, Dan had learned that each book had a copyright monitor that reported when and where it was read, and by whom, to Central Licensing. (They used this information to catch reading pirates, but also to sell personal interest profiles to retailers.) The next time his computer was networked, Central Licensing would find out. He, as computer owner, would receive the harshest punishment—for not taking pains to prevent the crime.
Of course, Lissa did not necessarily intend to read his books. She might want the computer only to write her midterm. But Dan knew she came from a middle-class family and could hardly afford the tuition, let alone her reading fees. Reading his books might be the only way she could graduate. He understood this situation; he himself had had to borrow to pay for all the research papers he read. (Ten percent of those fees went to the researchers who wrote the papers; since Dan aimed for an academic career, he could hope that his own research papers, if frequently referenced, would bring in enough to repay this loan.)
Later on, Dan would learn there was a time when anyone could go to the library and read journal articles, and even books, without having to pay. There were independent scholars who read thousands of pages without government library grants. But in the 1990s, both commercial and nonprofit journal publishers had begun charging fees for access. By 2047, libraries offering free public access to scholarly literature were a dim memory.
There were ways, of course, to get around the SPA and Central Licensing. They were themselves illegal. Dan had had a classmate in software, Frank Martucci, who had obtained an illicit debugging tool, and used it to skip over the copyright monitor code when reading books. But he had told too many friends about it, and one of them turned him in to the SPA for a reward (students deep in debt were easily tempted into betrayal). In 2047, Frank was in prison, not for pirate reading, but for possessing a debugger.
Dan would later learn that there was a time when anyone could have debugging tools. There were even free debugging tools available on CD or downloadable over the net. But ordinary users started using them to bypass copyright monitors, and eventually a judge ruled that this had become their principal use in actual practice. This meant they were illegal; the debuggers’ developers were sent to prison.
Programmers still needed debugging tools, of course, but debugger vendors in 2047 distributed numbered copies only, and only to officially licensed and bonded programmers. The debugger Dan used in software class was kept behind a special firewall so that it could be used only for class exercises.
It was also possible to bypass the copyright monitors by installing a modified system kernel. Dan would eventually find out about the free kernels, even entire free operating systems, that had existed around the turn of the century. But not only were they illegal, like debuggers—you could not install one if you had one, without knowing your computer’s root password. And neither the FBI nor Microsoft Support would tell you that.
Dan concluded that he couldn’t simply lend Lissa his computer. But he couldn’t refuse to help her, because he loved her. Every chance to speak with her filled him with delight. And that she chose him to ask for help, that could mean she loved him too.
Dan resolved the dilemma by doing something even more unthinkable—he lent her the computer, and told her his password. This way, if Lissa read his books, Central Licensing would think he was reading them. It was still a crime, but the SPA would not automatically find out about it. They would only find out if Lissa reported him.
Of course, if the school ever found out that he had given Lissa his own password, it would be curtains for both of them as students, regardless of what she had used it for. School policy was that any interference with their means of monitoring students’ computer use was grounds for disciplinary action. It didn’t matter whether you did anything harmful—the offense was making it hard for the administrators to check on you. They assumed this meant you were doing something else forbidden, and they did not need to know what it was.
Students were not usually expelled for this—not directly. Instead they were banned from the school computer systems, and would inevitably fail all their classes.
Later, Dan would learn that this kind of university policy started only in the 1980s, when university students in large numbers began using computers. Previously, universities maintained a different approach to student discipline; they punished activities that were harmful, not those that merely raised suspicion.
Lissa did not report Dan to the SPA. His decision to help her led to their marriage, and also led them to question what they had been taught about piracy as children. The couple began reading about the history of copyright, about the Soviet Union and its restrictions on copying, and even the original United States Constitution. They moved to Luna, where they found others who had likewise gravitated away from the long arm of the SPA. When the Tycho Uprising began in 2062, the universal right to read soon became one of its central aims.
See the page on gnu.org for more
The 2014 International Biocuration Conference [Byte Size Biology] 2013-10-17 14:51
For Ada Lovelace Day: Florence Nightingale [Byte Size Biology] 2013-10-15 10:16
Note: a repost of a 2010 post I published for Ada Lovelace day. Unfortunately, I am too busy these days to write a new one. “Ada Lovelace Day is celebrated today to “…raise the profile of women in science, technology, engineering and maths.”
So without further ado:
She is a ‘ministering angel’ without any exaggeration in these hospitals, and as her slender form glides quietly along each corridor, every poor fellow’s face softens with gratitude at the sight of her. When all the medical officers have retired for the night and silence and darkness have settled down upon those miles of prostrate sick, she may be observed alone, with a little lamp in her hand, making her solitary rounds
– the Times newspaper, 8 February 1855
It’s Ada Lovelace Day, and time to write about your favorite woman in science.
Florence Nightingale is best known for founding the profession of modern nursing. Today nursing is a skilled degree-earning profession, requiring extensive training, with professional rights and responsibilities. That was not the case less than 120 years ago, when normally only the military and religious orders offered semi-skilled assistance to physicians. Nightingale changed all that, and revolutionizing the way medicine is practiced. Historically known as the “Lady with the Lamp”, the angel of soldiers in the Crimean War, she ministered to the wounded not only with care and compassion, but with a newly-applied professionalism. This professional approach included keeping medical records and using them to improve health care.
Nightingale is less known for her managerial and statistical acumen, and her pivotal role in medical statistics. Nightingale kept meticulous notes of mortality rates at the Scutari hospital in Istanbul which declined dramatically during her administration. Upon her return to London, she compiled the records into a new polar diagram, known as Nightingale Rose Chart. The data is plotted by month in 30-degree wedges. Red represents deaths by injury, blue – death by disease, and black – death by other causes.
Note that this is not a pie-chart. The wedges are all in 30-degrees (so 12 wedges/months fill a circle) and the contribution of each cause of death is proportional to each wedge’s radius. Nightingale’s visualization of the role preventable diseases play in battlefield deaths made a very strong case to military authorities, Parliament and Queen Victoria to carry out her proposed hospital reforms. Specifically for adopting hospital sanitation practices and dramatically reducing death from preventable infectious diseases.
Here is an interesting critique of the Nightingale Rose Charts which is presented at Dynamic Diagrams. It appears that by placing the preventable diseases wedge-section in the outer section of the wedge, the blue received a proportionally larger area, an artifact of this radial plot. This does not detract at all from her achievements, and, as shown in the corrected charts, not even from her case for improving hospital sanitation to reduce preventable diseases as the leading cause of death, regardless of presentation format. (Pie charts are usually problematic).
You can read more about the Mathematical affiliation of Nightingale in this excerpt form the Newsletter of the Association for Women in Mathematics. One interesting factoid: she was the first woman to be nominated a fellow of the Royal Statistical Society.
Why not use the Journal Impact Factor: a Seven Point Primer [Byte Size Biology] 2013-10-11 16:10
What is the Journal Impact Factor?
The JIF is supposed to be a proxy for a journal’s impact: i.e. how much influence the journal has in the scientific community. It is calculated as follows:
A = number of times that articles published in the journal in years X-1 and X-2 from this journal were cited in year X
JIF(X) = A/B
Seems simple enough. The ratio of the number of citations to the number of publications. The higher the ratio, the more the articles are being cited. Therefore, the journal’s impact is higher.
Why is the JIF a bad metric? There are several reasons.

An editorial in Nature from 2005 stated that:
For example, we have analysed the citations of individual papers in Nature and found that 89% of last year’s figure was generated by just 25% of our papers.
The most cited Nature paper from 2002–03 was the mouse genome, published in December 2002. That paper represents the culmination of a great enterprise, but is inevitably an important point of reference rather than an expression of unusually deep mechanistic insight. So far it has received more than 1,000 citations. Within the measurement year of 2004 alone, it received 522 citations. Our next most cited paper from 2002–03 (concerning the functional organization of the yeast proteome) received 351 citations that year. Only 50 out of the roughly 1,800 citable items published in those two years received more than 100 citations in 2004. The great majority of our papers received fewer than 20 citations. (Emphases added my me).– Nature 435, 1003-1004 (23 June 2005)
Look at the equation describing how JIF is calculated. Note that the definition of B, the denominator, is “citable items”. If follows that the lower B is, the higher a journal’s overall impact factor. So the value of B is important for the overall impact factor. How, exactly, is the number of citable items in each journal determined? Yeah, good luck with answering that. From the editors of PLoS Medicine:
During discussions with Thomson Scientific over which article types in PLoS Medicine the company deems as “citable,” it became clear that the process of determining a journal’s impact factor is unscientific and arbitrary. After one in-person meeting, a telephone conversation, and a flurry of e-mail exchanges, we came to realize that Thomson Scientific has no explicit process for deciding which articles other than original research articles it deems as citable. We conclude that science is currently rated by a process that is itself unscientific, subjective, and secretive.
During the course of our discussions with Thompson Scientific, PLoS Medicine’s potential impact factor— based on the same articles published in the same year—seesawed between as much as 11 (when only research articles are entered into the denominator) to less than 3 (when almost all article types in the magazine section are included, as Thomson Scientific had initially done).
– The PLoS Medicine Editors (2006) The Impact Factor Game. PLoS Med 3(6): e291.
Many articles have two publication dates: upon acceptance, the article gets published in the journal’s online site. This is known as a “pre-publication” or “prepub”. The second date can be a few weeks or months later, when the publication coincides with the journal’s print date. The latter is often used as the “official” date of publication. However, the article has existed for a while before the official date, gaining readership and perhaps citations. An article by Tort and colleagues shows that this lag is a highly significant contributor to a journal’s JIF.
The JIF is commonly used as a measure by hiring committees, promotion and tenure committees and some grant review panels to predict a scientist’s success. The logic in doing so is as follows: if she publishes in a “good place”, then her research has merit, and will have impact in the scientific community. Furthermore, if she published in good places, she will continue to do so. Those assessing institutions have good reason to need a predictive measure: universities and grant agencies invest money in their faculty, and would like to know if these scientists would be successful. But the JIF is a poor metric for assessing, let alone predicting, an individual scientist’s impact. In fact, many scientific councils urge universities, institutes and grant agencies NOT to use the JIF for these purposes. Moreover, Thomson-Reuters themselves maintain:
In the case of academic evaluation for tenure it is sometimes inappropriate to use the impact of the source journal to estimate the expected frequency of a recently published article. Again, the impact factor should be used with informed peer review. Citation frequencies for individual articles are quite varied.

Some journals limit the number of citations that can be used in an article. This encourages a more subtle form of bad science writing practice. The author, faced with a limit on the number of possible cited items, would tend to cite review articles rather than the original research articles. The reason being that one review article may cover material in several research articles. Consequently, this inflates the number of citations review articles get, and “steals citations” from research articles. As a result, journals which publish review articles (either fully or partially) get a higher impact factor. As it is, review articles are more highly cited than research articles, but limitations on number of citations exacerbate this situation. The dynamic I described renders the JIF even more unreliable, favoring review articles over research articles.
Again, referring to Brembs’s paper: JIF is a statistical predictor for the retraction rate in a journal. In other words, the higher a journal’s JIF, the higher the frequency of papers that are retracted from that journal. Brembs does not report on why that is. There are probably several contributing factors: a high-profile publication gets to be read ans scrutinized more; competitors in the field may be “out to get you”. But also, there may be pressure to publish high and therefore “cut corners” when performing research. We don’t know whether retractions from high ranking journals are due to a higher fraction of poor papers than in low ranking journals, or whether that is because the papers in high ranking journals tend to be scrutinized more carefully be more people. Be that as it may, the correlation of JIF and retraction rate is disturbing.
Because of the strong incentives to publish in high-impact journals, researchers can sequester their findings for years, delaying actual communication of their research. It can take an extraordinarily long amount of time to publish findings in a high-impact journal. These journals have very low acceptance rates, so a paper can get delayed for years while the research and papers get revised and resubmitted over and over. Even worse, research would get tailored towards what is perceived as impactful and “sexy”. Another problem, raised recently by the editor in chief of Science, Bruce Alberts, is the overcrowding of fashionable fields:
But perhaps the most destructive result of any automated scoring of a researcher’s quality is the “me-too science” that it encourages. Any evaluation system in which the mere number of a researcher’s publications increases his or her score creates a strong disincentive to pursue risky and potentially groundbreaking work, because it takes years to create a new approach in a new experimental context, during which no publications should be expected. Such metrics further block innovation because they encourage scientists to work in areas of science that are already highly populated, as it is only in these fields that large numbers of scientists can be expected to reference one’s work, no matter how outstanding.
Caveat: I could not find research supporting these points, and I really only wrote it based on personal experience and conversations with colleagues. If anyone out there knows of supporting research (how would you even begin is an interesting question), let me know.
Concluding with an excerpt from an editorial by Kai Simmons, published in 2008, unfortunately still true today:
There are no numerical shortcuts for evaluating research quality. What counts is the quality of a scientist’s work wherever it is published. That quality is ultimately judged by scientists, raising the issue of the process by which scientists review each others’ research. However, unless publishers, scientists, and institutions make serious efforts to change how the impact of each individual scientist’s work is determined, the scientific community will be doomed to live by the numerically driven motto, “survival by your impact factors.”
Further reading:
Quit smoking, more bacteria will like you [Byte Size Biology] 2013-09-27 09:21
As an ex-smoker I can attest to this: quitting is hard. It can also make you fat. I gained quite a few kilos when I quit, and those took a long time to lose. Happily, these days I am spending money on running marathons rather than on cigarettes.

Got a light?
Weight gain after smoking cessation is fairly well known. In fact, fear of weight gain is cited as a primary reason many smokers are reluctant to quit. There’s a large body of research on weight gain in those who quit. In fact, one thought is that smoking cessation, when accompanied by weight gain, can throw the quitter from Scylla to Charybdis: glucose tolerance becomes lower, and you are now in danger of developing metabolic disease if you do not watch your weight gain. Therefore, intervention against weight gain is common practice in smoking cessation programs.
But why do smokers gain weight? Common answers are: nicotine is an appetite suppressant (and we even may know how); the metabolism of smokers is higher. Also, food is used as a replacement for smoking: quitters need that dopamine surge they are not getting anymore from nicotine. They are getting it from food, and especially the fatty, salty and sweet food that is so unhealthy, yet (like cigarettes) is designed to be addictive.

mmmm……….
But now, a group in Switzerland has found something new that may affect in post-cessation weight-gain: gut bacteria. Apparently, changes to the diversity and composition of gut bacteria are profound, and those changes seem to affect weight gain. Reminder: the bacterial composition of the gut flora of obese and lean people are markedly different. In a series of observations in humans, and experiments in mice, gut flora have been shown to directly affect weight gain. The type of bacteria that predominate in the gut of obese mice (and people) break down sugar more efficiently. Hence, a vicious cycle is created where an obese person would gain more calories from the same food than a lean person. Fortunately, the cycle can be broken through change of diet. And, as shown more recently, possibly by fecal transplant.
(Personally, I’ll stick with the change of diet, thankyouverymuchindeed.)
But back to those who quite smoking (yay!) but started gaining weight as a result (grrrr…). The changes in the phyla of their gut bacteria resemble those of obese people, although with some important differences, which may or may not mean that the gut flora changes are responsible for weight gain. For instance, the species in the smokers’ pre-quitting gut flora was fairly similar to that of a lean person, but much less diverse. That is an interesting finding by itself: somehow, smoking reduces gut flora diversity; but why that is, and what kind of effect this has on smokers’ health, is the subject for other studies. The post-quitting flora, on the other hand, was mostly similar to that of an obese person, with the difference that the flora was more diverse than the pre-quitting flora. taken together, these findings are pretty incredible: smoking causes such profound changes to our body, that the number and type of bacteria that settle in our guts are different between smokers and non smokers. And if you are a smoker, less bacteria “like” you: smoking somehow selects for certain types of bacteria. To me, the main finding was the increase in microbial diversity after quitting, not the gain of Firmicutes which may or may not contribute to post-cessation weight gain.
So: quit smoking. Even your bacteria will thank you. Eventually, a diverse gut is a healthy one.
Biedermann et al. Smoking Cessation Induces Profound Changes in the Composition of the Intestinal Microbiota in Humans (2013) PLoS ONE 8(3): e59260. doi:10.1371/journal.pone.0059260
Postdoctoral Position in Computational Bioengineering (Rice University) [Byte Size Biology] 2013-09-13 13:51
The ideal candidate will have a strong enthusiasm for interdisciplinary work. Required skills include excellent analytical skills, excellent software engineering skills, experience with programming in C++, and extensive exposure to biochemistry and biophysics. A Ph.D. in a related field is required for the position. Experience with Rosetta is highly desirable. In the past successful candidates have had degrees in Computer Science, Bioengineering, Biophysics, Physics, and Chemistry. Excellent communication and collaboration skills are required as the selected candidate will be expected to work closely with current lab members and with collaborators. To indicate interest please send a CV to Lydia Kavraki (kavraki@rice.edu). Applications will be accepted until the position is filled.
The Kavraki Group is a small but vibrant group and provides a stimulating working environment. Most of its alumni are now in academic or upper-level research positions. For more information about the group check http://www.kavrakilab.org. The Computer Science and Bioengineering Departments at Rice are part of the Gulf Coast Consortia for for Quantitative Biomedical Sciences and offer an unparalleled training environment (http://www.gulfcoastconsortia.org/home.aspx). Rice University is a leading research university in the vibrant urban setting of Houston, TX, the fourth largest city in the U.S. Rice is a small, private university with exceptional strengths in engineering, biomedicine, and nanotechnology. The Computer Science Department is among the top 20 in United States, while the Bioengineering Department is ranked among the top 10 departments in United States by US News & World Report.
Announcement: WikiProject Computational Biology Competition [Byte Size Biology] 2013-09-09 16:08
WikiProject Computational Biology/ISCB competition announcement 2013
The International Society for Computational Biology (ISCB) announces an international competition to improve the coverage on Wikipedia of any aspect of computational biology. A key component of the ISCB’s mission to further the scientific understanding of living systems through computation is to communicate this knowledge to the public at large. Wikipedia has become an important way to communicate all types of science to the public. The ISCB aims to further its mission by increasing the quality of Wikipedia articles about computational biology, and by improving accessibility to this information via Wikipedia. The competition is open to students and trainees at any level either as individuals or as groups.
The prizes provided by ISCB for the best articles submitted will be:
1st prize – $500 (USD) and 1 year membership to the ISCB.
2nd prize – $250 (USD) and 1 year membership to the ISCB.
3rd prize – $150 (USD) and 1 year membership to the ISCB.
For more information go to http://en.wikipedia.org/wiki/Wikipedia:WikiProject_Computational_Biology/ISCB_competition_announcement_2013
Contact Person: Alex Bateman (agb@ebi.ac.uk)
The Bio* projects: a history in graphs [Byte Size Biology] 2013-09-07 17:00
Yesterday I received an email from Kristjan Liiva, a student at RWTH Aachen University Germany. Kristjan has developed a really cool dashboard to analyze and visualize the development of collaborative OSS projects by mining their mailing lists and software repositories. (If the link doesn’t work, try again later; the project is heavily under development). The result is a very interesting picture of social trends in collaborative OSS projects.
Kristjan has mined the mailing lists and repositories of Biopython, Bioperl and Biojava, all three bio* (‘biostar’) projects have large developer and user communities, and have been around for over a decade.
One thing Kristjan did, is create a graph for each year, where the nodes are people, and the edges are based on email communications. You see a map of what biopython looked like in the early days, 2000:

Note Jeffrey Chang (then a graduate student at Stanford), Andrew Dalke and Brad Chapman (then a graduate student at University of Georgia, Athens) with >5 edges each. They were quite busy at the time.
Biopython got bigger the next year (2001):

Note the same actors are the “hubs”: Brad, Jeffrey and Andrew. Although they have more edges now, and there are new, local hub actors. Of note is Thomas Hamelryck , who wrote most of the structural biology part of Biopython. But he appears in two nodes, (as thomas@cbs and thomas ‘at’ cbs), so his contribution has been diluted in this graph. Many, many of people contributed, and some got cut off by my rendering, sorry.
Here’s 2004:

I was helping to roll releases, so I got kinda “hubbish” myself, with many edges on my node. A couple of years later I was looking for a new job, so I mostly dropped out of this scene.
The last year on Kristjan’s dashboard is 2011:

Peter Cock is the main active character in the graph (and he still is, and you are doing an amazing job Peter, BTW, probably not hearing that enough!) along with João Rodrigues, Brad Chapman, and Eric Talevich, among many others. Again, sorry about the cropped screenshot.
EDIT: if you go to the dashboard, and select “view by release” instead of “view by year” you can highlight the core contributors for each release. I assume that was done by number of contributions to the source versioning system.

Of course, there have many contributors over the years, and Biopython and the other bio* projects would not be so successful without all contributing users that provide such a diverse amount of code. Thanks to Kristjan for his work, and for letting me write about it. I’m looking forward to seeing this project develop. The social aspects of OSS projects are no less intriguing than the technological ones!
Are you using this blog for teaching, studies or writing? [Byte Size Biology] 2013-09-03 13:33
Dear readers (Yeah, I’m talking to both of you!)
If you are a school teacher, college professor or any kind of other educator, trainer or science writer, and if you have ever used this blog in your line of work, please let me know. Also, if you are a student and used this blog as a source, please let me know. I would like to assess the impact of this blog for several reasons, including grant proposals and for a general assessment of the outreach and community impact of my department.
So: if you have used this blog in any way in your line of work (reading this blog to avoid work does not count, unfortunately), please tweet me (@iddux), or email me: idoerg ‘at’ gmail ‘dot’ com or just comment on this post. Please include:
Name
Profession / employer
How many posts did you use (roughly) and for what purpose? For example: “high school teaching, 3 posts”.
Is it time to retire Pagel's lambda? [Lab Notebook - R] 2013-10-11 03:00
Pagel’s \(\lambda\) (lambda), introduced in Pagel 1999 as a potential measure of “phylogenetic signal,” the extent to which correlations in traits reflect their shared evolutionary history (as approximated by Brownian motion).
Numerous critiques and ready alternatives have not appeared to decrease it’s popularity. There are many issues with the statistic, some of which I attempt to summarise below.
The \(\lambda\) statistic is defined by the Brownian motion model together with a transformation of the branch lengths: multiply all internal branches by \(\lambda\). The motivation for the definition is obvious: \(\lambda = 1\) the tree is unchanged and the model equivalent to Brownian motion, while for \(\lambda = 0\) the tree becomes a star phylogeny and the model is equivalent to completely independent random walks. \(0 < \lambda < 1\) provides an intermediate range where the correlations are weaker than expected.
Problem 1: It is biological nonsense to treat tips different from other edges.
All other problems arise from this. While it is okay that a statistic does not have a corresponding evolutionary model, being part of an explicit model might have helped avoid this sillyness. Technically \(\lambda\) is a model, but one that treats evolution along “tips” as special, as if evolution should follow completely different rules for a species alive today relative to it’s former evolutionary history. Sounds almost creationist.
Problem 2: The statistic doesn’t measure what is says it measures.
To demonstrate this, we can consider two cases in which phylogeny has the identical effect of explaining trait correlations, and yet have very different lambdas. Consider that Researcher 1 examines the phylogeny in Figure 1 and estimates very little phylogenetic signal, \(\lambda = 0.1\).
{r} library(ape) cat("(((A_sp:10,B_sp:10):1,C_sp:11):1,D_sp:12);", file="ex.tre", sep="\n") ex <- read.tree("ex.tre") plot(ex)
Now Researcher 2 discovers closely related sister species of some of the taxa originally studied, as in Figure 2.
{r} cat("((((A_sp:1, A2_sp:1):9,(B_sp:1, B2_sp:1):9):1,(C_sp:1, C2_sp:1):10):1,(D_sp:1, D2_sp:1):11);", file = "ex2.tre", sep = "\n") ex2 <- read.tree("ex2.tre") plot(ex2)
There traits of sister taxa are very similar (indeed let us assume the sister species are hard to distinguish morphologically - perhaps why they were overlooked by Researcher 1). The OU or BM model estimates made by researcher 1 will closely agree with with those of Researcher 1, since the sister taxa have quite similar traits. Yet the \(\lambda\) estimates differs greatly – all of a sudden the phylogenetic signal must be quite high!
And yet the underlying evolutionary process by which we have simulated the data has been unchanged! The difference arises because what formerly appeared as long tips have become short tips. How do we intepret a metric that depends so heavily on whether or not all sister species are present in the data? As noted, this problem does not impact other phylogenetic comparative methods to nearly the same extent.
Problem 3 The statistic has no notion of timescale or depth in the phylogeny.
In \(\lambda\) (and other definitions such as Blomberg’s K), phylogenetic signal is an all-or-nothing proposition. If we find that really recently diverged species that happen to resemble each-other, while species that have diverged for longer than, say, a couple million years show no correlation – is this phylogenetic signal or not? This ‘extinction’ of phylogenetic signal as we go far enough back in time seems like a biologically reasonable concept that is perfectly well expressed in the \(\alpha\) parameter of the OU model, but is lost in the consideration of \(\lambda\). If folks really want to estimate a continuous quantity to measure phylogenetic signal, I suggest \(\alpha\) is a far more meaningful number (note that it has units! (1/time or 1/branch length)).
Consider the returning force alpha in the OU model (i.e. stabilizing selection). When alpha is near zero, the model is essentially Brownian, (i.e. ‘strong phylogenetic signal,’ where more recently diverged species are more similar on average than distantly related ones). When alpha is very large, traits reflect the selective constraint of the environment rather than their history, and so recently diverged species are no more or less likely to be similar than distant ones (provided all species in question are under the same OU model / same selection strength for the trait in question). The size of alpha gives the timescale over which ‘phylogenetic signal’ is lost (in units of the branch length). Two very recently diverged sister-taxa may thus show some phylogenetic correlation because their divergence time is of order 1/alpha, while those with longer divergence times act phylogeneticly independent, such as in our Figure 2 above. I find this an imperfect but reasonable meaning of phylogenetic signal.
If we restrict \(\lambda\) to be strictly 1 or 0 these problems are alliviated, though then it is unnessary to define the statistic as such as we may instead consider a star tree (sometimes called the “white noise” model of evolution).
Pagel’s \(\delta\) is a transformation on node depth, which is again problematic as there is no meaningfully consistent way to describe what is a node (think about deep speciation events with no present day ancestor.) I believe \(\kappa\) would also be problematic to interpret as it is a nonlinear transform of branch length – raises branch length to a power – and thus would have a rather different effect depending on the units in which branch length were measured. (For instance, consider the case where the tree is scaled to length unity, so all branch lengths are less than one and thus become shorter with large exponents, vs one in which lengths are all larger than one). Fortunately these statistics are far less popular than \(\lambda\).
Reflections on the Mozilla Science Code Review Pilot [Lab Notebook - R] 2013-09-25 03:00
I was recently interviewed a Nature senior reporter Erika Check Hayden on the subject of the scientific code review project being conducted by Mozilla Science Lab. The piece appears in this week’s issue, Hayden 2013. My blog post sharing my own approach to code review is mentioned at the beginning of the article, though it is rather Roger Peng’s comments at the end that have stirred some interesting discussion.
Roger raises two concerns. First, that increased scrutiny will discourage researchers from sharing code, (which, right or wrong, remains a voluntary choice in most journals):
One worry I have is that, with reviews like this, scientists will be even more discouraged from publishing their code
and second, that code review does not focus on what matters mosts:
We need to get more code out there, not improve how it looks
(Erika provides a bit more context to Roger’s comments below).
@ctitusbrown @cboettig @kaythaney @nickbarnes see whole @simplystats quote on prof. code review discouraging sharing pic.twitter.com/pNQWT9Safz
— Erika Check Hayden (@Erika_Check) September 25, 2013
The Nature News piece thus nails a central tension in the community between promoting higher standards for code (A position exemplified in an earlier Nature column titled “Computational Science … Error: Why scientific programming does not compute”, and more recently in Science by Joppa et al. 2013, which explicitly calls for peer review of scientific software) vs promoting more widespread sharing of software (as exemplified by Nick Barnes piece in the same issue, “Publish your computer code: it is good enough”.
The arguments made in each of the perspectives are excellent, and should be required reading for anyone interested in the subject. In addition to the shorter comment by Barnes, I also recommend the more recent Nature perspective, The Case for Open Computer Programs, which lays out the argument and modest practical recommendations (that have largely been ignored as far as I can tell) just brilliantly. In particular, I think they nail the issue of why describing the algorithm or providing pseudo-code is not a satisfactory description of the method.
However, I also think the tension between review and sharing is somewhat artificial. While each of these positions emphasizes the need to share source-code, the call for code review by Merali, Joppa et al (or in my own blog post mentioned earlier), focus on scientific software aimed at reuse by others. The concerns voiced in Roger Peng’s comments and echoed by Nick Barnes focus on another class of code entirely – code associated with a particular research publication that would primarily serve only to document and support those results, rather than be readily adapted to other uses.
@cboettig @ctitusbrown @kaythaney but far more important to get code out than to get it “right”, IYSWIM.
— Nick Barnes (@nickbarnes) September 25, 2013
For me, the crux of these concerns lies in the difference between “software papers”1 and papers which merely use code in some element of the methodology. The Mozilla study focused exclusively on code appearing in the full-text of publications in PLoS Computational Biology. Though I am pleased to see the Science Lab tackle the issue of software review and bring the expertise of their professional software developers to bear on scientific code, this is perhaps not the kind of code I would have chosen to focus on (something I shared with the team early on in seeing the announcement).
Without knowing which papers are included it is of course difficult to say to much. But knowing that the code appears in the full text of the papers themselves, we can assume that it is not a complete software package intended for reuse by other researchers. Using code within the body of a manuscript implies the intent to communicate methodology more concisely and precisely than might be done in prose; in much the same manner that we use equations in place of prose. This is an important development in scientific communication, but is also rather distinct from the use of code in other contexts, in which the code itself is meant to be read primarily by machines. It is code that is already intended to help explain.
Code included in appendices to scientific papers is meant rather to document exactly what has been done, in a manner that assists replication, and may require considerable effort to decipher exactly what is being done. Instead, it merely supports the more readable but less precise description and potentially the pseudo-code that would appear in the body text.
Code intended for reuse as research software (in software papers) is another class entirely. Ostensibly, the user never needs to see the code itself, but only interact with the user interface or end-user functions (API) provided. Code that is written clearly and concisely still has value – helping identify bugs and facilitating future researcher-developers extending the software, but most of it’s functionality can be accessed and assessed without looking at the source. I think it is in this kind of review that we as a researcher-developer community could learn the most from the Mozilla software engineering experts.
I believe the most important focus of code review is in scientific software rather than in code snippets. And in reviewing software, I think all of the most important elements do not actually involve reading the source code at all (as I discuss in my revised position on reviewing software papers), but rather in establishing that the software behaves as expected and follows software development practices that make it more sustainable, such as hosting in a software repository, version control, or example input and output.
Roger’s second comment appears more dismissive of code review than I think it actually is:
“We need to get more code out there, not improve how it looks.”
@kaythaney @ctitusbrown yup, ‘pretty’ is dismissive terminology, though possibly short-hand for ‘human-readable’ not just ‘machine-readable’
— Carl Boettiger (@cboettig) September 25, 2013
Most modern languages include syntactic sugar: ways of expressing commands that are more easily interpretable to human readers. For instance, in C, a[i] is syntactic sugar for *(a+i). Higher-level languages are in some ways all sugar around existing lower-level libraries.2 Like good mathematical notation or good prose, this is not just about being ‘pretty’, but being more effective in communicating with humans. Certainly this is something we can improve upon as researchers, but it is perhaps not the best starting point.
If code review should apply to all levels of code or be reserved for scientific software may still be an open question. What we should be able to agree on is in the publishing of code in the first place:
@cboettig @kaythaney @nickbarnes I think code sharing should be mandatory shrug. It's part of the methods. I reject papers w/o it.
— Titus Brown (@ctitusbrown) September 25, 2013
It continues to surprise me how few journals require code deposition. Science explicitly adopted a new policy in 2011 stating
Science is extending our data access requirement listed above to include computer codes involved in the creation or analysis of data
which asks that the data be placed in a appropriate permanent repository or otherwise placed in the supplementary materials (see information for authors).
Yet I have not seen code provided for any analysis I have read in Science since 2011. Either we have a very different understanding of what it means to use computer codes in the analysis of data or Science grossly neglects its own policy.3 Not to pick on them of course, few other journals have explicitly adopted such a policy.
.@cboettig @kaythaney @nickbarnes As one of my grad students said to me, “I don't understand why ‘must share code’ is a radical opinion.”
— Titus Brown (@ctitusbrown) September 25, 2013
Nick Barnes suggests that this alone may be enough to improve code quality:
@cboettig @ctitusbrown @kaythaney require sharing. Pride will then rapidly lead to review and other improvement techniques.
— Nick Barnes (@nickbarnes) September 25, 2013
Certainly it will help, though not enough if the state of open source scientific software is any indication (Nick does acknowledge a rather geological notion of ‘rapid’). Smaller codes used in particular analyses will certainly feel this pressure more, as they will be easier to scrutinize.
Focusing on code appearing in-line in papers certainly addresses a different beast than large scientific software packages intended for reuse. As Roger observed, we will likely learn that scientists aren’t software engineers. We may learn how to use code to communicate more effectively. We may learn some lessons that apply for larger codebases involved in scientific software, but I think there the problem are often outside of the individual lines of code themselves and arise from other development practices.
Still, learning how to do code review at all would be an invaluable start. As the discussion on my own post on the subject made clear, we researchers have no training in this practice. The Mozilla study would give the first taste. I only hope they turn there attention next to larger scientific software that lives outside of the publications themselves and is intended at re-purposing and reuse. Meanwhile, I also hope journals will become more serious about recognizing code as methods, as they have started to do with data.
which I define as papers primarily aimed at promoting the reuse of a particular codebase and providing an indexed citation target to credit the work.↩
It is frequently argued that ideal code should be ‘self-documenting’, with syntax so precise that no other explanation of the function is necessary. While I think this is an admirable ideal, it should never be a substitute for actually providing prose documentation as well. I find we too easily decieve ourselves as to just how self-documenting our own code really is (it’s all so obvious at the time, right?).↩
From my reading it appears that Science requirements are intentionally too vague on this issue, stating: “All computer codes involved in the creation or analysis of data must also be available to any reader of Science.” It is unclear if ‘available on request’ is considered appropriate for code, though it is explicitly not acceptable for data: “we have therefore required authors to enter into an archiving agreement, in which the author commits to archive the data on an institutional Web site, with a copy of the data held at Science”, and the editorial makes it clear that “the data access requirement … includes computer codes”. Meanwhile all studies involving ‘requests for data’ have shown a return rate of substantially less than a 100% (usually less than 20%, e.g. this recent study). If that was a viable option we could just publish abstracts and have papers available on request too. If you need to know who wants your data, why not do the same for papers?↩
Forking The R Journal [Lab Notebook - R] 2013-07-10 03:00
All I really wanted to do is cite a paper in the R Journal, which is peer-reviewed, open access (CC-BY), LaTeX based and without author charges. Sure, I could do that already, but I like being able to programmatically generate citation metadata from a URL – we do have this thing called the internet now, and citations are just links, right? Unfortunately, nice as it is, the R journal doesn’t have HTML landing pages for articles that embed the metadata.
This makes it harder for Google Scholar to index the articles, and means that we cannot extract citation metadata from the URL using a tool like greycite. Until now.
I wrote to Editor-in-Chief Hadley Wickam about this, who responded in the best way possible: making the journal website’s Github repository public. A fork and a little hacking later, and voila, we have html landing pages for the R Journal with embedded metadata (pending a pull request). Check out the source code for how this works – it’s really quite straight forward since the metadata is already available in _config.yaml. The main step involves a Generator plugin which builds a page for each article and makes the relevant article metadata available to the page. Then we can write a page template using Liquid code to import the metadata.
The really exciting thing about this is the basic idea of forking a journal website and improving it. There’s a lot we could do to improve the R journal. I think that ideally we’d have HTML5 versions of the articles as well, something that would be straight forward if authors used knitr of course, but I realize that’s a bigger shift. One could also automatically enhance the HTML with quite a bit more semantic content, we could have animations, links the Rnw files, etc etc. I’d be happy to help and I’m sure others would too. R-journal is great but it could be so much more as an example and test-bed of innovation in statistical publishing.
Well, now that the journal is open for pull requests, hack away!
Reviewing Software Revisited [Lab Notebook - R] 2013-07-09 03:00
Several weeks ago I wrote a post describing issues I commonly raise when reviewing software papers. This provoked a lively discussion among authors, editors, and other reviewers of software papers (most of us having been on at least both reviewing and authoring end), with quite varied perspectives as to whether such criteria were appropriate in this context. As I describe there, I view the concept of dedicated software papers as a somewhat necessary “hack” of the publishing system, and hope a more mature system will eventually come into place, as is now happening for data through efforts such as Dryad and associated journal archiving requirements.
Sustainability of the software is important for the same reason archiving the literature is important – without this, we cannot build on existing work. Building on “researcher/developer” software (e.g. the kind of software typically covered in ‘software papers’) is currently an uncommon and risky, as eloquently argued by Brian O’Meara in the comments of an earlier post describing just such a failure.
Through that discussion, I’ve come to see the goal of reviewing software papers as one of software sustainability. After all, the publication becomes a part of the researcher’s permanent record and part of the permanent scholarly archive, designed to outlive the journal itself. It seems reasonable that the software it describes should at least exist and do something five years later. Though this sentiment underlies most of my criteria, my phrasing is often too specific and a little too grounded in language explicit to software development.
As I learned from Neil Chue Hong (Software Sustainability Institute) in a follow up from that discussion, The Journal of Open Research Software has recently revised its guidelines making much of this more explicit and much better worded than I did. They provide separate lists for the reviewer to comment on both the paper and the software – making it clear that the review goes beyond the paper. The questions for the paper are every bit as valuable as those from the software section: for instance, reference to sections on “Reuse”, “Quality Control”, and “Implementation and Architecture”. While these things might not get their own section headings in an MEE paper, the ideas should certainly be addressed. I think these guidelines provide an excellent checklist for promoting sustainable software without putting undo emphasis on questions of performance, scalability, extensibility, etc.
(content below CC-BY, quoted from Journal of Open Research Software)
Please provide a list of your recommendations, indicating which are compulsory for acceptance and which are optional but would improve the quality of the paper or the reusability of the software.
Like my own list, a repository, a license, and sample input-output data are all mentioned explicitly. Software review section suggests there should be a mechanism in place to “identify whether the software is operating as expected” – such as basic unit tests. Extensibility is also mentioned, but without any reference to features that may or may not assist in that – a fault of my own list, which was probably over-specific on that account. I think these are reasonable and solid guidelines, and would love to see other journals that frequently publish software papers (Looking at you, MEE, JSS, Bioinformatics, R Journal, PLoS Comp Bio) adopt similar criteria.
Of course I’d love to hear what others think about this.
What I look for in 'Software Papers' [Lab Notebook - R] 2013-06-13 03:00
Update Thanks to the rich discussion in the comments and beyond, I’ve revised my thoughts on this somewhat, as I discuss in this more recent post.
I am more and more frequently reviewing ‘software papers:’ which I define as publications whose primary purpose is to publicize a piece of scientific software and provide a traditional research product with hopes that it will receive citations and recognition from other researchers in grant and job reviews. To me this feels very much like hacking the publication recognition system rather than the ideal way to recognize and track the role of software in research communities, but a very practical one in the current climate. I have written two myself, so I have been on both ends of this issue. In this post, I share what I look for in such papers and what omissions I see most frequently.
If we are going to employ this hack of the publication system for software, we should at least use it to maximal advantage. As a reviewer, I feel that means reviewing not just the submitted manuscript but the software itself. If we can agree on nothing else, we as a community should at least be able to say:
my review of a software paper is a review of the software
I assume most other authors, reviewers and editors on this content share this implicit assumption, but I’d love to hear first hand from anyone else. For instance: as an editor, what would you do if your reviewers only commented on the paper directly and not on the software distributed?
We are not really taught to review software, any more than we are taught to write it in the first place. Most journals offer little guidance on this (though see the Journal of Open Research Software guidelines for peer review of software, all though they are still rather minimal.) In the absence of a culture on software reviewing, I thought I would lay out my own perspective with the hope of hearing feedback and push back from others. Perhaps through such dialogs we can develop clearer expectations for this rapidly expanding genre.
I don’t include “to document” the software as a purpose, since none do so very comprehensively, and besides, documentation belongs in the software, not in a journal. “Publicize” usually includes some motivating examples that could convince many readers that the software does something useful for them without too much effort. As such, I expect the paper to:
This is an intentionally low bar that I hope helps promote these kinds of contributions. Despite this, papers frequently fail the copy-paste test or the plain text explanations of the function calls. Come on people. Meanwhile, I try to focus the rest of my review on the software itself.
As I am almost always reviewing R packages, the software already meets some very basic standards required by submission to CRAN: dependencies and license stated, built-in documentation, passing some minimal automatic checks, etc. (See the CRAN Policies and the Writing R Extensions Manual for details). This is great, as it clears the first few hurdles of installation, etc. without much fuss, but still provides a bar that is by itself unacceptably low for published scientific software. Here is a list of the things I see that most often frustrate me. This isn’t intended as a style-guide or a comprehensive list of best practices, just my own pet peeves. I have somewhat tongue-in-cheek labeled them by severity of the review I might give; which like any other use of these terms is more of a measure of how annoyed I am then anything else. Critiques and suggestions welcome.
The comments from other reviewers, authors, and editors have been fantastic, thank you all. I particularly appreciate the opportunity to have reviewing styles critiqued, something that does not happen in normal peer review.
Just a note on my headings here. I do not see any of these things as “gatekeeping requirements” and have intentionally omitted the option of “Reject”. I would reject such a paper for methodological flaws, etc., but not for any of the reasons on my list below. The list is intended only to improve, not prevent, software publication.
I believe any of the decisions below typically result in a revision to the same journal, that authors judiciously choose how to respond to reviewer comments guided by the editor’s own feedback, and that it is ultimately the editor’s decision whether any of this is relevant. </edit>
A scientific R package must must must have some automated tests that are run by R CMD check. Even if further development of the package doesn’t break anything (most likely only if further development doesn’t happen), changes to the package dependencies could still break things, and so it is crucial to have a mechanism in place to detect these problems when they arise. For code that runs quickly, the simplest way to do this is through the examples in the documentation. I don’t expect all scientific software to have a complete test suite with 100% coverage covering all the weird things that can happen if a user passes in a matrix when the function expects a data frame or has some unanticipated missing values, etc. Just some tests to make sure the basic examples execute and I’ll be happy. Longer running functions or those that need calls to external web resources shouldn’t be run in the examples (too much of a burden for CRAN’s automatic testing) so they should be marked dontrun and put in a separate test suite or vignette as it says in the manual.
I see authors write functions like this all the time:
f <- myfunction(f, p){
# stuff
o <- optim(f, p)
# stuff
}
calling an existing library function like optim that has a whole host of very useful optional arguments that have a significant impact on how the algorithm functions. Whenever you a rich function like optim, please have the courtesy to make it’s arguments available to future users and developers through your function call. Yes, most users will just want the default arguments, (or your default arguments, if different), and that can be handled just fine by providing default values as optional arguments. R has a fantastic mechanism for this exact issue: the ... argument. The above code could be fixed simply by using:
f <- myfunction(f, p, ...){
# stuff
o <- optim(f, p, ...)
# stuff
}
which works just they way you think it would. If you have more than one such function (ask yourself if you can write shorter functions first and then) pass optional arguments as lists,
f <- myfunction(f, p, optim_options, fn2_options){
# stuff
o <- do.call(optim, as.list(c(f, p, optim_options)))
# stuff
b <- do.call(fn2, fn2_options)
# stuff
}
arguments can also be extracted with list(...)$arg1 etc.
A converse of this issue is not providing default arguments where it might be natural to do so. This does not bother me so much, as it is probably useful to force the user to think how many iterations n are appropriate for their problem rather than just assuming that 100 is good because it is the default. The only time this case is annoying is when the argument will not be changing – such as a user’s authentication token to access a web resource. Don’t make me manually pass the token to every function in the library please.
I would really like to see a link to the software development page, such as r-forge or Github. The primary asset in this context is pointing reviewers to an address with a bug tracking system where issues can be assigned ticket numbers and readers can transparently see if a package is being actively maintained. A reader who comes across the paper years later who has only an email address that may or may not work has little way to determine what the latest version of the code is, whether it is actively maintained, or whether earlier versions that may have been in used in previous publications suffered from any significant bugs.
We write software papers with the sometimes vain hope that they will be cited by users, so authors of such papers should at least follow these best practices themselves. R includes a native mechanism for providing citations to packages, citation(packagename), including the information for any software paper published along with it. Be sure to add your own software papers to the CITATION file. More information can be found in my post on Citing R packages.
These are other things that commonly frustrate me, but fall on a bit more of a continuum of style rather than gross oversights. As such I’m not sure that any one of these things would justify rejection.
Style guides will tell you to keep functions short, not more than a screen or 20 lines. Breaking large functions into a series of smaller functions and documenting those smaller functions – even if they are only used internally – is a great help to a reviewer trying to understand what a function is supposed to do and also test that it does what it says. Anyone building the code base later (most often yourself) will appreciate the reusable modules.
An extension of providing optional arguments to functions is to also provide access to all of their return information. To extend the example from wrapping optim, this would involve returning the convergence information. Using object classes and helper functions for return objects helps keep code stable and lets users leverage existing code for similar objects, such as fitting or plotting routines. More discussion on this topic based on my own experiences in the post, we need more object oriented design in comparative methods
Because CRAN requires this through the DESCRIPTION file, R package authors rarely neglect this entirely. A sometimes misconception is that because R itself is primarily dual-licensed under GPL-2 and GPL-3 that R packages must use a GPL license due to the “viral” clause of the GPL. This clause only applies if you are modifying existing GPL functions directly and is not a requirement for R packages, which recognize a large array of licenses. My own recommendation for authors seeking to maximize the impact of their work is to use MIT, BSD (2 clause), or CC0 license for the package. CC0 has the advantage of being suitable for and data or documentation included, but authors should do there homework and decide what is best for them.
Consistent use of coding style, good documentation, clear examples, intelligent reuse of code, and other best practices are all areas in which any work could improve. While we can all become better developers by highlighting these issues in our reviews, they are probably best developed over time and in dialog with the user community. I also put anything extending the scope of functionality into this category – I do not have any concept of minimal contribution as long as the code meets the criteria above. Meanwhile, there’s always a few pet peeves I just cannot help mentioning. Here’s one which is particular to R packages and so commonly overlooked.
Many developers overlook that package dependencies that provide functions your functions will use internally should be listed as under IMPORTS rather than DEPENDS. This keeps the users namespace cleaner and avoids collisions of functions having the same name. Use DEPENDS only for those packages whose functions will be used by the end user as well.
If you are an author, editor, or reviewer of R software packages, what are your pet peeves?
manuscript reviews on github? [Lab Notebook - R] 2013-06-10 03:00
I was recently impressed to learn of Trevor Bedford’s strategy of seeking pre-approval for posting his reviewer’s comments as Github issues. Beyond providing links to the data and source-code, I generally don’t advertise the open science nature of papers I submit – I guess I assume that if the reader or reviewers care, it should be easy enough for them to discover it. Consequently I am usually immediately frustrated to realize that upon receiving my reviews I have to create a second, private repository for the review material, our replies to reviewers, etc., as I don’t have permission to disclose that information. 1 I have recently stumbled across several examples of authors publishing to the web anonymous reviews they have received. Though anonymous, I feel the practice potentially murky without explicit permission, so I would appreciate any insight others have on this.
Trevor’s approach suggests I should consider broaching this question when first submitting my review, so I am puzzling over the best way to do so. One option would be to include such a request in the cover letter. For example,
The authors of this manuscript would like to request that the editor and reviewers indicate in their replies if they consent or decline to have their comments posted anonymously in the public Issues Tracker of this paper.
Does that need more explanation? A link to examples like Trevor’s that have done this before? Do I need to explain the value of having this kind of transparent provenance for the paper? Should I mention how this could give the reviewer more transparent credit? Encourage them to comment directly on Github from their own account?
Does this need the blessing of the journal? How would you feel about such a clause as a reviewer or editor? For a recent review I had done of a paper that was similarly written on Github, I obtained the Journal’s permission to post my review as an issue in their repository. I would love to see more examples of this kind of thing, if anyone has come across them.
While I lean towards a minimal statement such as the one above, perhaps a cover letter would be a good place to document some of the other open and reproducible features of the manuscript? Or perhaps such statements should be added to the manuscript itself? Among the options, I might point out:
make pdf in the repository directory.Listing all of these would make for a somewhat lengthy cover letter, which might be a bit overwhelming to be useful (or seem more promotional than valuable). Are any of the seven things above worth highlighting in particular?
Perhaps these details could be deferred to a README file in the project’s Github repo, and the cover letter could simply provide a link to the project repository? What, if anything, would appear most useful and accessible to a reviewer unfamiliar with this approach or its potential value? Though elements of this approach have been discussed in the published literature, e.g. Gentleman & Temple Lang (2007) ‘compendium’ concept, Stodden (2009) RRS concept, or Peng (2011) reproducible papers in the Journal of Biostatistics, I’m unsure if pointing a reviewer to these references would be more valuable or more confusing. Let me know what you think.
Strictly speaking the edits to the manuscript in the open repository could also be considered confidential, though at that stage I haven’t yet signed the copyright agreements that come with publication, which tend to be quite reasonable even for the traditional subscription based journals I work with↩
DOI != citable [Lab Notebook - R] 2013-06-03 03:00
I feel I see this kind of comment almost daily:
Is there a way to obtain DOI for a @github repository? (for citing #opensource software packages, similar to @figshare objects) #git
— Ahmed Moustafa (@AhmedMoustafa) May 29, 2013
Again and again, researchers suggest that DOI to makes something “citable”. And this frustrates me.
Don’t get me wrong. I love DOIs, and I love CrossRef. And I bang on the table when I have some old journal article that doesn’t yet have a DOI. I use DOIs every day in many ways. I use CrossRef’s APIs all the time to draw in metadata for citations in my notebook (through my knitcitations package), and to import metadata into my reference manager, Mendeley. I’ve written my own implementations in R and ruby, and keep an eye on their exciting new tools on the Crossref Github page. I wrote to bibsonomy when I realized they were not using the CrossRef API to look up metadata by DOIs, and they have now implemented this feature. I use DOIs to look up papers I’ve come across, and to share content I am reading. (Crossref’s DOI shortener is great for this). I even use DOI-based links to embed semantic information into links and citations of articles.
But I still have no idea what researchers mean when they suggest that this makes something citable.
At its heart, a DOI is a very simple concept. It is a “permanent identifier”. All this means is that is is really just a URL redirect. Type http://dx.doi.org/mnn into any browser and get redirected to where the article actually lives. Why does that make it permanent? Because if the journal decides to change their URL structure, the DOI’s redirect can just be mapped to the new address and voila, it still works. That is, a DOI is simply a tool to fight link-rot.
So you might ask, why does the ability to remap the address have anything to do with being “permanent?” It doesn’t, really. The permanence comes not so much from the technology as from the social contract that goes with it. As CrossRef’s Geoffery Bilder eloquently explains, a publisher can only receive DOIs if they promise to keep these redirects up-to-date. A publisher who fails to maintain this responsibility would presumably lose their right to receive DOIs. A brilliant, simple, social incentive.
This still does not guarantee permanence – e.g. what would happen to the content if the publisher disappears. That problem is not addressed by the DOI technology itself, but by a robust backup archiving solution, such as CLOCKSS, which provides a geo-politically distributed network of backup copies for many journals. Again the social contract comes into play – presumably CrossRef would not provide a publisher with DOIs if they did not have such a robust archival solution in place.
So far we have seen two crucial functions of the DOI – as a permanent identifier that can be used to reach the content despite link rot, and as an incentive to maintain good archival backups of the content and the links to it.
So what does this have to do with being citable? Obviously these are nice properties to have for things we cite – but they are by no means a requirement. (As Noam Ross observes, try finding a permanent identifier for “Personal Communication”). Books, reports, and other grey literature frequently appear in citations, as do links to websites. MLA even has guidelines on the proper format to cite a tweet (which, incidentally, come closer to having a permanent identifier and an archival strategy than most other things in this list). So what do we mean by citable anyway?
But what about the reference list? While a publisher may be just fine including some link to your software, is it really cited if it isn’t in the reference list? Journals restrict what appears in the reference list because these references are indexed by the infamous citation counters like Thompson-Reuters. (A frequent complaint is that many journals do not similarly index citations appearing in the reference list of the supplementary materials, making it difficult or impossible to give appropriate attribution to large numbers of data providers, for instance). Does having a DOI address this problem?
Counting citations depends on who is counting them. The most well-known is Thompson-Reuters, which has their own process for deciding what gets counted (based on publisher), so no guarantee there. Meanwhile Google Scholar counts anything meeting it’s indexing requirements & arbitrary selection. I have recently learned that CrossRef just launched it’s own internal citation counting, which is available from the CrossRef metadata (totals only for the public, publishers can resolve which articles did the citing…). However, most proposals to make some alternative research product “citable” by giving it a DOI use DataCite DOIs (e.g. figshare, PeerJ Preprints), which lag behind in this feature. Moving the control of citation data beyond the grasp of particular publishing companies like TR is undoubtedly an important step forward. The Open Citation Project is a more comprehensive, if very young, move in this direct. (Hat tip to Martin Fenner for explaining CrossRef citations to me).
In addition to resolving links, DOI providers also serve a rich collection of metadata about the publication that can be queried by DOI or by other elements like author and title. Rich semantic formats and disambiguation of author names by connections to ORCID IDs are among the many advantages of this. Because many of these tools are publicly accessible by through their APIs, it is easy for other developers to build services upon them.
While DOI providers have done an excellent job in ensuring persistent URLs, archived content, and valuable metadata, these things are largely the product of the social contract between publisher and the DOI provider. It is not possible for an author or organization to simply “get DOIs” for all their content. But it is not the only way to provide these features, either. While I understand the value in providing a simple and reliable way to encapsulate each of these concepts as “has a DOI,” it also appears to put these features beyond the reach of individual researchers. If issues of persistent URLs, archived content, and rich metadata tools are always reduced to “has a DOI,” publishers become the only path to achieve these ends. On the contrary, a rich collection of tools is available to researchers.
So what do we mean when we say a DOI makes something ‘citable?’ If this is shorthand for the properties we would want in something citable: persistent identifier, archival content, machine-readable metadata, than we should start to recognize other things that share these features. Further innovation requires valuing the features the DOI provides, not simply a “brand name” researchers recognize.
In a recent post in a series on technical features of my open notebook, I discuss some of the tools available to address these challenges. In particular:
Of course, if you ever need a DOI for a research product, there is always figshare.
Notebook features: digital archiving [Lab Notebook - R] 2013-05-31 03:00
Note: this entry is part of a series of posts which explain some of the technical features of my lab notebook.
Archival preservation of digital scholarly content is an important challenge throughout the research community. As the notebook is the permanent record of my regular scholarly endeavors, this provides the opportunity to experiment with tools and techniques for digital archiving while also better preserving the notebook. In the experimental spirit that drives all my exploration here, I am testing several ways to accomplish this. In so doing, I learn which approaches are easiest to implement, to use, and gather feedback from, while also hedging my bets in the event that any given strategy should fail.
Archiving digital content involves two fundamental challenges that can be difficult to satisfy simultaneously: providing a robust backup copy of the content, and providing a consistent location (such as a URL) where the content can be retrieved.
The simplest archival measure employed in the notebook comes from hosting through my own domain, carlboettiger.info rather than an external server. By controlling the domain structure myself, I am not tied to a University server that can be altered by an IT department without my knowledge, thereby breaking my links. When I choose to move platforms, as I did in migrating from Wordpress to Jekyll, I could ensure that links would be appropriately mapped. This was not the case when I started my open notebook on the OpenWetWare platform, since links are all mapped to the openwetware.org domain which I obviously cannot control, even though I could at least migrate my content. HTML redirects make sure links still resolve when I change structure (e.g. carlboettiger.info/archives/211). I don’t have to worry about moving my domain when I change institutions, and can seamlessly migrate to a different server or DNS provider to get better rates or uptime performance.
Of course these advantages are also the greatest weaknesses of this approach – they all depend entirely on me. I could make or forget to make any number of changes that could cause this all to break. Time has shown that even the best-intentioned researchers are not the best curators of there own data, and no doubt I am no exception. How can the content and its identifying addresses outlive me or my interest in it?
PURLs, or Persistent Uniform Resource Locators, provide a DOI-like mechanism for addressing the challenge of link-rot. As Geoffrey Bilder eloquently argues, the technological solution is quite simple, the real challenge lies on the social side of the implementation – a social contract that promises content providers will maintain their identifiers if they want to continue to receive identifiers. Though users must register to be able to create PURLs, PURL does not provide such enforcement.
The PURLs solution is a bit more web-native solution than DOIs, in being more democratic, using a URL structure, and being built upon widely distributed servers and open-source web technology. Not surprisingly, other web-native systems such as most of the major semantic web ontology providers rely on PURLs, e.g. Dublin Core uses purl.org/dc/terms/. The PURL FAQ provides a great overview.
Implementing PURLs for the notebook was very straight-forward. After registering as a user at purl.org I applied for my own top-level domain: cboettig, which I then mapped to my current domain, carlboettiger.info By enabling partial redirects, each page on my site will also be resolved using this top-level domain followed by my existing page structure. Following my existing structure is not necessary – I could map each page to an arbitrary path in my domain, but would have to enter these somewhat manually. While the partial redirect is simpler to implement, it does require that I maintain the rest of the link structure.
In this way, purl.org/cboettig/lab-notebook.html now resolves to carlboettiger.info/lab-notebook.html. Likewise, each page in the notebook can be similarly resolved from the purl.org domain instead of my personal carlboettiger.info domain. Should I ever somehow lose control of carlboettiger.info, I could re-assign my PURL to redirect to my new domain URL. This provides DOI-like technology of permanent identifiers for every page in the notebook.
Committing content to an external repository is the recommended way to avoid link-rot from the user errors and website changes that so frequently plague self-archiving of scholarly content. Keeping multiple copies of content in geographically distinct locations is the time-honored approach of digital archiving. Git and GitHub make this easy. Not only does this mean that a backup copy is publicly available and forkable online, but it is also easy to clone copies on each of the machines I work on and rely on git to keep them in sync. Should Github disappear, a little git remote add and everything will be effortlessly deployed with complete history elsewhere.
The notebook has two Github repositories: the “source-code” consisting of plain-text (markdown) content and Jekyll-based templates on labnotebook, and a second for the rendered HTML cboettig.github.com (which also now hosts the website).
While a custom domain and PURLs provide persistent locators for the content, distributed copies on Git help archive the content itself. Should my domain vanish or Github disappear, copies of the content, complete with version history, would remain distributed across various machines with a copy of the repository. Links to Github would break in that process, unless we had remapped all links from the notebook to Github using PURLs.
I think of good metadata as the third leg to proper digital archiving, in addition to permanent identifiers and backup of content. We want to be able to point a tool at the permanent identifier / URL of an entry and extract reliable information on the author, time published and last modified, title, author, key words, etc. that might be useful in citing or categorizing the content. Providing this information is really the subject of adding Semantic metadata to the site, and is covered in another entry in this series. Meanwhile, the Greycite tool and it’s API are an excellent way to extract this metadata into a variety of useful formats, working much the same way that CrossRef’s tool does using DOIs. Here is an example query
Depositing a copy of the notebook on figshare is one of the most robust archival solutions of which I am currently aware. Not so much because it has the coveted DOI solution to the permanent identifier problem but because it has the promise of CLOCKSS archiving, should anything ever happen to figshare.
Nevertheless, it raises several challenges. The native home for the content is as rendered HTML at my domain, not as raw HTML on an archive completely unassociated with that domain, difficult to view, and divorced from my usual workflow, unlike my usual publishing source-code to Github and website to my domain. It also raises questions of just what to archive and when. I discuss some of these strengths and challenges as a separate post, archiving the lab notebook on figshare: advantages and challenges.
Digital archiving is a challenging problem that is not completely addressed by any one of these approaches. In the end, robust archiving will be best left in the hands of its experts. Unfortunately, the best examples currently available (such as CLOCKSS, national libraries, etc.) will not archive a researcher’s web page directly. The solutions explored here are not perfect, but they are free and simple to implement. I’d love to hear what others think.
Archiving the lab notebook on figshare [Lab Notebook - R] 2013-05-31 03:00
One of the most comprehensive approaches I have come across so far uses figshare. This offers the most promising avenue for content preservation, but is weakest in managing the URIs and associating them with the original content. All figshare content is archived by CLOCKSS, an international library cooperation providing redundant and geopolitically distributed backup of the archives around the world (and used by many academic journals, both subscription based & open access). Should figshare vanish from the face of the planet, it will trigger the release of all of its content to resolve through the CLOCKSS servers, with the same appearance and resolving at the same URLs as the original figshare content. Presumably the DOIs provided to figshare content will also continue to resolve there.
It would certainly be preferable to have the notebook archived by CLOCKSS directly, since the association between the original online content at carlboettiger.info is lost in archiving the entries on figshare. More problematically, the content as archived on figshare is not recognized by search engines, etc., as a separate HTML pages to index, but merely as a bundle of attached text files. On the upside, the content becomes part of the global scientific datasets preserved and indexed by figshare with appropriate metadata, etc., increasing the chances for discovery through that venue. Also, figshare provides a convenient API that can help automate deposition.
Deciding just what to archive in the figshare database is also less straight forward than it may seem. I have gone through a few iterations:
_site HTML included (?)I began by archiving the markdown files that I write to create each entry. These are plain-text files that can be easily read in any text editor, even if the conventions for rendering them as HTML are lost. Like HTML, figures are linked to external files, and are thus not captured by this solution. To work around this, I adopted the trick of using Data URIs to embed images. This places a binary encoding of the image in the text itself, which can be rendered by almost any browser as the appropriate image. While this keeps the content together, the long data URI strings are rather out-of-place inside a plain text document. Further, the markdown loses all of the valuable semantics that are automatically added to each page when Jekyll renders them to HTML. As Phil Lord argues, if there’s any format that future archivists can read successfully – it will be HTML. Consequently I’ve settled on archiving the HTML versions of each notebook entry, with images embedded as Data URIs. Each HTML file contains rich metadata in the header, sidebar, and footer, that give more information about the content and its context in the notebook (relative path, categories and tags, timestamps and SHA hashes, etc). I have archived these entries in annual chunks following the year/month/day directory structure already employed on the site.
There is still additional external content used to render the site – CSS and javascript files – that are not captured in this approach. Though entries actually render just fine without the css, it would certainly be possible to include this material in the archive (though some Javascript comes from external CDNs). This does make for a bit larger and more cluttered archive, and more to the point is a rather crude solution to a problem already solved by Internet archiving programs such as CLOCKSS or internetarchive.org.
Lastly there is the concern of preserving the version history of entries. Though figshare provides versioning of its content, this doesn’t capture finer resolution of individual page changes available through the Github repository. At the expense of creating an ever more cumbersome archival object, one could include the .git history, either for the HTML rendered version (which lives at cboettig.github.com) or the source files used to create it (labnotebook).
Of course this fails to address the preservation of externally linked content. The most frequent outbound links point to other publications through, usually their DOIs, which we hope will take care of themselves. The most important externally linked content in the notebook entries are the links to scripts, functions, and manuscripts in the various project repositories on Github. The simplest solution is to embed the most important scripts in the notebook entries themselves. Archiving the project repositories is an additional challenge, but if a user can recover a copy of the project repository (along with it’s .git history) then it would be possible to identify the linked file using the SHA hash from these links (by matching it against the SHAs in the log). See my entry on SHA hashes for more on this topic.
Current and previous archives of my lab notebook can be found on figshare by year. Older versions of these archives have taken a different approach, including just archiving the markdown files. The links use the DOI and point to the most recent version. (At this time linking to explicit versions with FigShare’s DataCite DOI links doesn’t appear to be working)
Notebook features: SHA Hashes [Lab Notebook - R] 2013-05-03 03:00
Note: this entry is part of a series of posts which explain some of the technical features of my lab notebook.
I version manage all changes to my entry using git. Each page is linked to its source history on Github, which will display a list of all previous edits to the post with an easy-to-read commit log and highlighted diffs. A version history is often considered an essential part of an open lab notebook, where changes to the notebook are documented and preserved. While wikis, Google docs, Dropbox, Wordpress plugins, or just regular backups can provide version history of pages, none come close to comparison with a full version management system such as git. This is because git’s underlying architecture is based on
The magic of cryptographic SHA hashes.
Hashes provide an immutable and verifiable record of any all changes I make. Because the hash is generated by the cryptographic SHA1 algorithm from the contents of the site, it is impossible to make changes without causing the hash to update. By referencing the content’s hash value we can be sure to link to a constant version of the entry. These can be verified by re-generating the hash (requiring the previous state of the repository in this case, see note below). Unlike publication timestamps, versions, or DOIs, this provides a way not only to reference particular versions of a file, but a cryptographically secure way to verify that the version is what it claims to be. Tobias Kuhn has observed that this is a valuable feature we should want to see for all scientific publishing. Each of my posts now displays its SHA hash on the sidebar along with other metadata. While the history button already provides a convenient way to browse all previous versions of a post, I chose to display the SHA hash directly so that the hash value would be part of the document metadata, while also highlighting this feature.
In addition to the GitHub repository for my lab notebook, My research code, analyses, and manuscripts are collected into Github repositories by project. This allows my analysis and paper writing to benefit from this same immutable and verifiable record. Because GitHub uses the SHA hashes in its link structure, this also provides a convenient way to link to a particular version of code in a given entry. This way, I can be sure the contents of the file displayed at that link never change, even as I continue to update that file. Even if the file or containing directory is later deleted or moved, the link will still resolve. Only if the entire project repo were deleted or if Github itself dissolved would the link be lost. Even then, using the SHA hash given in the link we could determine the contents of the file from some other copy of the repository (such as a local or figshare archive).
Tobias is actually working on his own SHA hash approach which is somewhat superior to the simpler method of using git. The Github hash corresponds to the state of the entire repository/notebook at the time of the commit, rather than the contents of an individual file. Consequently, one would need a snapshot of the entire repository, available on Github, to perform the verification. Tobias is looking into generating hashes based on the contents of the file directly – so far, only RDF data – that could provide a unique and verifiable reference for any scholarly data or publication.
Version managing the notebook and code has many more practical day-to-day benefits, such as recovering from a mistaken deleted or corrupted file, merging changes made on different machines or by collaborators, or creating branches to test new features without disrupting current version, and comparing differences as a file evolves.
Scholarly Infrastructure Thoughts [Lab Notebook - R] 2013-05-02 03:00
Happened across a provocative example of why we need a software ecosystem, though it was certainly not intended to be one, which led me to ask myself:
How complex does an algorithm have to be before a talented researcher with expertise in both the relevant mathematics and computer science will make a significant mistake in their first release of the software?
As a corollary we might also ask, “How much less care do we put into research code not destined for release?”
This changelog clearly reflects these difficulties face well-established researchers with long publication records on these very methods. If such individuals can make mistakes in packages, are we supposed to trust the myriad personal implementations of this and more complex algorithms that bulwark our literature today?
Academic knowledge is currently built in the mortar and bricks of publication and citation. Publications advance new claims built up on existing claims (and very occasionally replacing them) through citations. It an approach that does not scale well on many fronts. To verify information we must trace the citation chain, which grows far to quickly to for human processing and is not usually amenable to computer processing. Yet here it is the statistical scaling that concerns me more – a paper advances that a claim is true with a certain probability, given that the methods are implemented without error. The more publications we string together, the higher the probability that we observe false positives, but also that we observe implementation errors. For much of research today, we need not construct the scientific argument in this manner.
Thanks to computational advances of the last several decades, public repositories of the data and the methods (such as can be implemented as software anyway), we can build on existing work by direct analysis of the data and methods, rather than treating the conclusions as given. Evidence would no longer come primarily in the highly circumstantial manner of citations to previous claims, but to direct analysis of data. Using a common pool of data and methods would align incentives better to maintain and improve upon this infrastructure (the way major companies contribute to the underlying ‘plumbing’ provided by shared open source infrastructure), while there is no incentive for the literature to work in such a way.
Musings On Conservation Literature [Lab Notebook - R] 2013-05-02 03:00
Just because I’m a theorist deeply entrenched in methodological concerns about uncertainty and decision making doesn’t mean I don’t think about practical conservation from time to time. Some musings from my comment here, copied over for my own reference.
Though I would like to believe the gap stems from the problems you discuss, I think that differing objectives between research and application may play a much larger role. I suspect that scientific papers that are most useful and influential for conservation practitioners and policymakers are those which confirm what they already believe, or whatever the interests opposed to them least want to hear. Let’s call these “Cassandra” papers, since in this context they usually forecast disaster. For the practitioner it may matters little whether the math is simple or complex, clearly explained or impenetrable, or even right or not so right. Worm et al 2006 paper which the media quickly decided predicted the end of global fisheries within 50 years is perhaps a good example.
Okay, so beyond bolstering arguments already being made by those who propose, implement or legislate conservation against their opposition, there are certainly unknowns that they might turn to research to answer. Resource allocation might be an example of this; e.g. do we prioritize purchasing pristine areas that are not likely to be threatened or less pristine areas in more immediate danger (a la Pfaff). Let’s call these “rule of thumb” papers, where the conclusion is an easily applied guide-line. It seems doubtful that the practitioners would be inhibited by their access and understanding of the math in this case, since they want the research to provide an answer they can trust, and not worry so much about what math justifies it. They are more likely to use proxies of quality (journal, researcher, affiliation, popularity of the method), then working through the assumptions to see if they like them; no?
So there is a third case in which the conclusion is of the sort “apply my method and it will tell you what to do”, as opposed to “here’s what to do”. I think only this case falls at risk to the mathematics being a barrier, though when accompanied by user-friendly software tools perhaps that can be dismissed as well. These “methods” papers are probably the favorite option of many researchers, as they seem the most rigorous, accurate way, reflecting the details of the problem at hand. Scientists are probably least fond of the first example, where even the paper’s authors may feel the conclusions are being overstated, while others feel such work is wrong and counterproductive. I’d guess many researchers are lukewarm towards the middle case, as better than a coin flip. I imagine from the conservation practitioner’s ranking is reversed. To what extent would you agree with this classification? If so, how is the conservation literature distributed across these categories, and how might we want it to be distributed? Do we indeed have the greatest impact writing Cassandra papers rather than writing nice clear methods, and if so, what are the implications?
Notebook features: reproducible code [Lab Notebook - R] 2013-04-26 03:00
I now use the dynamic documentation software called knitr to write most entries that shore results and figures. The code to replicate the results is included automatically, ensuring that what I say I did and what code I actually ran to get the results are consistent. Though I have written about knitr before, both regarding its use in my notebook and in my manuscripts, here I provide a quick summary of how a reader might actually reproduce a figure or result they come across in the notebook, as well as some of the possible problems involved.
As the code required for any given analysis can be quite involved, it is not pratical to provide free-standing scripts in this way. Instead, I write the algorithms as functions provided by an R package dedicated to the project, e.g. nonparametric-bayes, multiple-uncertainty, or warning-signals, which is version-managed on Github. The code displayed in the post can then be limited to the specific calls to analysis, data manipulation, and plotting functions unique to the exploration shown, without repeating the code for all algorithms involved.
The code for the analyses is also stored on github using the same dynamic documentation approach with knitr. These scripts are found in the inst/examples directory of my packages. This approach allows a given analysis to evolve with my research in a more tractable way than simply pasting updated copies as successive notebook entries. The notebook entries then become a place for me to synthesize the results of a script.
Though the package functions are usually backwards-compatible, proper reproducibility is only attained by having the version of the package from time of the result. This is easily accomplished by the seemless integration of Github and R using the devtools package. Consider a figure from a page of the notebook, such as the final histogram plot from this entry. The entry links to the script responsible for the figure, https://github.com/cboettig/nonparametric-bayes/blob/9d5cd1f027bdfe5f356dce83756726c95a6fcdd8/inst/examples/myers-exploration.md
We can install the entire research compendium at exactly the state it was at the time of the analysis using the hash (long chain of seemingly random characters, see the (upcoming) entry on hashes) using the clever devtools R package,
install_github("nonparametric-bayes", "cboettig", "9d5cd1f027bdfe5f356dce83756726c95a6fcdd8")
We can then copy and paste the code linked from the figure to replicate the results. This provides a fast and effective way replicate the work appearing in or linked to any entry. More importantly perhaps, this approach also allows one to repeat any given analysis with the most recent version of an algorithm and compare the results, since the package structure provides a logical seperation between algorithm and analysis. In practice such fine-grained control and invistigation is more important than simply being able to regenerate what has already been done without any further input.
This is not entirely failsafe. The package may depend on other packages, which themselves may have changed. For my use cases, it is a deal more reliable than running the current version of a package that is actively changing during my research. Readers interested in even more robust replication and verification should take a look at Roger Peng’s package Eckel S and Peng RD (2012). stashR: A Set of Tools for Administering SHared Repositories. R package version 0.3-5.“>Eckel & Peng (2012)stashR package and associated publications
Notebook features: an introduction [Lab Notebook - R] 2013-04-26 03:00
In keeping this open lab notebook, I have sought to address three goals (in addition to all the traditional reasons for keeping a lab notebook)
which are coincidentally evocative of NSF’s Broader Impacts areas. In this series of posts I plan to explore and illustrate some of my experiments to address these goals through various web-based tools available for an open notebook platform. Many of these have been documented in the notebook itself as I experiment with them (see #notebook-technology). Not all of those experiments pan out and older tools and techniques are often replaced with newer ones as I explore, and these posts are usually more technical notes written to help me think through and remember what I’m trying out. In order to provide a more accessible snapshot of notebook features, I thought it might be helpful to write a series of posts describing these tools and techniques.
Below is an index of posts in this theme that I will continue to update as I have a chance to finish them. If there’s anything about the notebook that you’d like to hear more about, feel free to suggest it in the comments.
Notebook Features: parsing linked data in the semantic notebook [Lab Notebook - R] 2013-04-04 03:00
In a post a while back I originally put forward the idea of a semantic lab notebook. Semantics, or linked data, are among the most powerful concepts to emerge in online science for scholarly data organization and communication. I have slowly been adding and exploring new ways to introduce semantic concepts into the notebook, which I have documented along the way under my #semantics tag. In this post, rather than discuss how to generate the semantic data, I try to focus on some of the things we can do with it. This really just scratches the surface of possibilities, but should at least illustrate the general idea.
We will start with some very simple examples exploiting the semantics inherent in HTML5. We can then work up to richer examples that rely on XML-based parsing. The richest potential of the semantic notebook lies in leveraging the RDFa content, whose terms are defined as ontologies over which a machine can apply reasoning and formal logic against other web content (see, e.g. Shadbolt et. al. (2006) for further unformation). Though we show how to extract the and parse the RDF here, exploiting the RDF in the last example must wait to a later post.
Our first set of examples will address parsing the semantic HTML directly. For background on how these elements are added to the notebook, see this entry. We will use R with it’s excellent collection of web, XML parsing, and text-mining tools to take advantage of the semantic structure. First we load the required packages,
library(RCurl)
library(XML)
library(tm)
library(wordcloud)
library(RColorBrewer)
We can get a list of pages to download from the sitemap
pagelist <- readLines("http://carlboettiger.info/sitemap.txt")
pagelist <- pagelist[grep("/201\\d/", pagelist)] # drop non-posts)
pages <- lapply(pagelist, getURLContent, followlocation = TRUE)
Or, if an archive is available locally, (e.g. from figshare cache), we can read the files in directly.
pages <- system("ls ~/Documents/labnotebook/_site/2***/*/*/*.html",
intern = TRUE)
We parse each of the pages as HTML so that we can manipulate them with XML tools
html <- lapply(pages, htmlParse)
For instance, we can easily get the content of all entries:
text <- sapply(html, xpathSApply, "//article", xmlValue)
We can also extract metadata based on the semantic markup.
titles <- sapply(html, xpathSApply, "//title", xmlValue)
categories <- sapply(html, xpathSApply, "//node()[@class='category']",
xmlValue)
tags <- sapply(html, xpathSApply, "//node()[@class='tag']", xmlValue)
R makes it easy to summarize this data, e.g. by generating a table of the number of entries in each category, or a wordcloud of the tags.
table(unlist(categories))
computation ecology evolution open-science teaching
40 376 287 85 17
wordcloud(Corpus(VectorSource(tags)))
Citation information can be encoded
We can perform more direct text mining as well. For instance, we extract all DOIs found in the text:
doi_pattern = "\\b(10[.][0-9]{4,}(?:[.][0-9]+)*/(?:(?![\"&'<>])\\S)+)\\b"
require(gsubfn)
dois <- strapply(text, doi_pattern, perl = TRUE) #text[-462]
head(sort(table(unlist(dois))))
10.1002/(SICI)1520-6602(1998)1:1 10.1002/bjs.6880
1 1
10.1002/etc.2140 10.1006/jtbi.1998.0660
1 1
10.1006/jtbi.2000.1080 10.1006/jtbi.2001.2299
1 1
Or generate a wordcloud of the full text
carl <- Corpus(VectorSource(text))
carl <- tm_map(carl, removePunctuation)
carl <- tm_map(carl, tolower)
carl <- tm_map(carl, function(x) removeWords(x, stopwords()))
carl.tdm <- TermDocumentMatrix(carl)
carl.m <- as.matrix(carl.tdm)
carl.v <- sort(rowSums(carl.m), decreasing = TRUE)
carl.d <- data.frame(word = names(carl.v), freq = carl.v)
wordcloud(carl.d$word, carl.d$freq, scale = c(8, 0.4), min.freq = 3,
max.words = 100, random.order = FALSE, rot.per = 0.15, colors = brewer.pal(8,
"Dark2"))
RDF triples are the mainstay of semantic, linked data. Unlike the more text-mining oriented examples above, data in this format follows a strict and universal standard which allows a machine to identify meaning rather precisely. Critically, this allows one to automatically link data appearing in the notebook to data elsewhere on the web without the ambiguities of natural language that for instance, might confuse the animal jaguar with the car.
RDFa is a way of adding these precise statements to HTML, again see the earlier entry on how this is done. The technically inclined will note that the namespaces of the RDFa itself are not accessible in the XML parsing we used above, since they do not correspond to nodes or attributes, but appear only in the values of attributes. Fortunately, there are many excellent tools to extract this RDFa data, turning it into the XML formatted RDF triples we need. e can perform this using the Any23 API
download.file(paste("http://any23.org/rdfxml", "http://carlboettiger.info",
sep = "/"), "temp.xml")
doc <- xmlParse("temp.xml")
Which creates a beautiful RDF XML file of all linked data found in the entry.
doc
<?xml version="1.0" encoding="UTF-8"?>
<rdf:RDF xmlns:xhtml="http://www.w3.org/1999/xhtml/vocab#" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#">
<rdf:Description rdf:about="http://www.carlboettiger.info/">
<dcterms:title xml:lang="en">Carl Boettiger</dcterms:title>
<xhtml:license rdf:resource="http://creativecommons.org/publicdomain/zero/1.0/"/>
<dcterms:title xml:lang="en">Carl Boettiger</dcterms:title>
<dcterms:creator xml:lang="en">Carl Boettiger</dcterms:creator>
<dcterms:date xml:lang="en">2013-04-04T11:07:14-07:00</dcterms:date>
<dcterms:format xml:lang="en">text/html</dcterms:format>
<dcterms:language xml:lang="en">en</dcterms:language>
<dcterms:identifier xml:lang="en">/index.html</dcterms:identifier>
<dcterms:rights xml:lang="en">CC0</dcterms:rights>
<dcterms:source xml:lang="en">Lab Notebook</dcterms:source>
<dcterms:subject xml:lang="en">Ecology</dcterms:subject>
<dcterms:type xml:lang="en">website</dcterms:type>
<title xmlns="http://ogp.me/ns#" xml:lang="en">Carl Boettiger</title>
<author xmlns="http://ogp.me/ns#" xml:lang="en">http://www.carlboettiger.info/index.html#me</author>
<first_name xmlns="http://ogp.me/ns/profile#" xml:lang="en">Carl</first_name>
<last_name xmlns="http://ogp.me/ns/profile#" xml:lang="en">Boettiger</last_name>
<published_time xmlns="http://ogp.me/ns/article#" xml:lang="en">2013-04-04T11:07:14-07:00</published_time>
<site_name xmlns="http://ogp.me/ns#" xml:lang="en">Lab Notebook</site_name>
<url xmlns="http://ogp.me/ns#" xml:lang="en">http://www.carlboettiger.info/index.html</url>
<type xmlns="http://ogp.me/ns#" xml:lang="en">website</type>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info#me">
<rdf:type rdf:resource="http://xmlns.com/foaf/0.1/Person"/>
<rdf:type rdf:resource="http://schema.org/Person#Person"/>
</rdf:Description>
<rdf:Description rdf:about="http://www.carlboettiger.info/assets/img/carlboettiger.png">
<depiction xmlns="http://xmlns.com/foaf/0.1/" xml:lang="en"/>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info">
<homepage xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://carlboettiger.info"/>
<url xmlns="http://schema.org/Person#" rdf:resource="http://carlboettiger.info"/>
<name xmlns="http://xmlns.com/foaf/0.1/" xml:lang="en">Carl Boettiger</name>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info#me">
<jobTitle xmlns="http://schema.org/Person#" xml:lang="en">a post-doctoral researcher</jobTitle>
</rdf:Description>
<rdf:Description rdf:nodeID="node17eprp1n4x899515">
<rdf:type rdf:resource="http://xmlns.com/foaf/0.1/Person"/>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info#me">
<knows xmlns="http://xmlns.com/foaf/0.1/" rdf:nodeID="node17eprp1n4x899515"/>
</rdf:Description>
<rdf:Description rdf:nodeID="node17eprp1n4x899515">
<homepage xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://users.soe.ucsc.edu/~msmangel/"/>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info#me">
<knows xmlns="http://xmlns.com/foaf/0.1/" rdf:nodeID="node17eprp1n4x899515"/>
</rdf:Description>
<rdf:Description rdf:about="http://users.soe.ucsc.edu/~msmangel/">
<name xmlns="http://xmlns.com/foaf/0.1/" xml:lang="en">Marc Mangel</name>
</rdf:Description>
<rdf:Description rdf:nodeID="node17eprp1n4x899516">
<rdf:type rdf:resource="http://xmlns.com/foaf/0.1/Person"/>
<homepage xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://swfsc.noaa.gov/staff.aspx?&id=17294"/>
</rdf:Description>
<rdf:Description rdf:about="http://swfsc.noaa.gov/staff.aspx?&id=17294">
<name xmlns="http://xmlns.com/foaf/0.1/" xml:lang="en">Steve Munch</name>
</rdf:Description>
<rdf:Description rdf:about="http://carlboettiger.info#me">
<affiliation xmlns="http://schema.org/Person#" rdf:resource="http://boe.ucsc.edu/~msmangel/CSTAR.html"/>
<workplaceHomepage xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://www.ucsc.edu/"/>
<weblog xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://www.carlboettiger.info/lab-notebook.html"/>
</rdf:Description>
<rdf:Description rdf:nodeID="node17eprp1n4x899517">
<rdf:type rdf:resource="http://schema.org/PostalAddress"/>
<address xmlns="http://schema.org/Person#" xml:lang="en">Center for Stock Assessment Research, 110 Shaffer Rd, Santa Cruz, CA 95050, USA</address>
<streetAddress xmlns="http://schema.org/PostalAddress/" xml:lang="en">Center for Stock Assessment Research, 110 Shaffer Rd</streetAddress>
<addressLocality xmlns="http://schema.org/PostalAddress/" xml:lang="en">Santa Cruz</addressLocality>
<addressRegion xmlns="http://schema.org/PostalAddress/" xml:lang="en">CA</addressRegion>
<postalCode xmlns="http://schema.org/PostalAddress/" xml:lang="en">95050</postalCode>
<addressCountry xmlns="http://schema.org/PostalAddress/" xml:lang="en">USA</addressCountry>
</rdf:Description>
<rdf:Description rdf:about="https://orcid.org/0000-0002-1642-628X">
<orcid xmlns="http://purl.org/spar/datacite/" rdf:resource="https://orcid.org/0000-0002-1642-628X"/>
</rdf:Description>
<rdf:Description rdf:about="http://www.carlboettiger.info#me">
<rdf:type rdf:resource="http://xmlns.com/foaf/0.1/Person"/>
<account xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="http://www.cloudflare.com/email-protection#f497969b9180809d93b49399959d98da979b99"/>
<account xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="https://twitter.com/cboettig"/>
<account xmlns="http://xmlns.com/foaf/0.1/" rdf:resource="https://github.com/cboettig"/>
</rdf:Description>
<rdf:Description rdf:about="http://www.carlboettiger.info/">
<license xmlns="http://www.carlboettiger.info/" rdf:resource="http://creativecommons.org/publicdomain/zero/1.0/"/>
<license xmlns="http://creativecommons.org/ns#" rdf:resource="http://creativecommons.org/publicdomain/zero/1.0/"/>
</rdf:Description>
</rdf:RDF>
We can now explore this data using the XML tools illustrated above. The rigidity of the XML rather than HTML parsing and the use of namespaces gives us greater precision.
Semantic Citations For The Notebook And Knitr [Lab Notebook - R] 2013-02-22 03:00
I have on ocassion been exploring the use of semantic markup in the notebook. In this post I illustrate how I am handling semantic citations. One of the more intriguing ideas is the ability to add semantic meaning to citations through the CITO ontology of Shotton (2010). Citation counts form a central part of academic discourse, but contain very little information regarding the reason for the citation. Most notably, ‘negative’ citations refuting a claim carry just the same weight as those confirming or relying upon a claim. Given the scale and expansion of academic literature, it is rarely reasonable to explore this citation graph manually. CITO provides a language for embedding the meaning of the citation, such as “discusses”, “refutes”, or “usesMethodIn”, to the citation. (For instance, my earlier citation to Shotton identifies itself as “usesMethodIn”, as I will explain).
The main barrier to this approach is a lack of adoption. One of the primary concerns is the burden it places on authors of adding the extra data. On one hand, authors already bother formatting and reformatting layout, spelling, and reference order to the arcane specifications of different journals, which suggests authors can be persuaded to do some pretty tedious tasks if the publishers would require it. After all, the task of adding citations is already much easier than it was in the days of paper journals. Still, it is much simpler to remove a tedious requirement than to add a new one. My hope is that intelligent tools can simplify this process, as they already have with other aspects of managing citations, and encourage the use of CITO. In this spirit, I have recently started trying to consistently use the CITO ontology in my notebook entries as a test case, using some tools of my own design.
Several months ago I created the R package knitcitations to provide a citation platform for knitr dynamic documents, which provide executable code and automatic inclusion of results inside plain-text (markdown) descriptions. I write most of my research scripts and many of my notebook entries in this manner. The package can generate citations by DOI, circumventing the need for maintaining bibtex or similar database of citation information, using commands such as
citet("10.1186/2041-1480-1-S1-S6")
Extending the package to support CITO was rather straight forward. Using the latest version of knitcitations, one can generate in-line citations with CITO semantics simply by passing the reason for the citation as well, such as
citet("10.1186/2041-1480-1-S1-S6", cito="usesMethodIn")
which generates the following HTML:
<a href='http://dx.doi.org/10.1186/2041-1480-1-S1-S6' property='http://purl.org/spar/cito/usesMethodIn' >Shotton (2010)</a>
This provides a convient platform to generate semantic citations in this lab notebook. As before, knitcitations will also generate a complete reference list at the end of the document by calling the bibliography function at the end.
It is possible to add far more semantic data to this reference list at the end of an article. Invisible semantic markup can identify to a machine what value corresponds to the volume number or issue number, or journal name, e,g, using the BIBO ontology. I have added support for ths kind of markup to knitcitations as well, and several of my posts provide examples. The raw markup looks like this:
<div prefix="dc: http://purl.org/dc/terms/,
bibo: http://purl.org/ontology/bibo/,
foaf: http://xmlns.com/foaf/spec/,
biro: http://purl.org/spar/biro/"
rel="http://purl.org/spar/biro/ReferenceList"> <ul class='bibliography'>
<li> <span property="dc:title">Fisheries: Does Catch Reflect Abundance?.</span> <span property="dc:creator"> <span property="foaf:givenName">Daniel</span> <span property="foaf:familyName">Pauly</span>, </span><span property="dc:creator"> <span property="foaf:givenName">Ray</span> <span property="foaf:familyName">Hilborn</span>, </span><span property="dc:creator"> <span property="foaf:givenName">Trevor A.</span> <span property="foaf:familyName">Branch</span>, </span> (<span property="dc:date">2013</span>) <span rel="http://purl.org/dc/terms/isPartOf"
resource="[http://purl.org/dc/terms/journal]">
<span property="http://purl.org/dc/terms/title"
content=" Nature ">
</span>
<span property="bibo:shortTitle"> Nature </span>
</span> <span property="bibo:volume">494</span> <a property="bibo:doi" href="http://dx.doi.org/10.1038/494303a">10.1038/494303a</a> </li>
<li> <span property="dc:title">Net Gains.</span> <span property="dc:creator"> <span property="foaf:givenName">unknown</span> <span property="foaf:familyName">unknown</span>, </span> (<span property="dc:date">2013</span>) <span rel="http://purl.org/dc/terms/isPartOf"
resource="[http://purl.org/dc/terms/journal]">
<span property="http://purl.org/dc/terms/title"
content=" Nature ">
</span>
<span property="bibo:shortTitle"> Nature </span>
</span> <span property="bibo:volume">494</span> <a property="bibo:doi" href="http://dx.doi.org/10.1038/494282a">10.1038/494282a</a> </li>
</ul>
</div>
However, I have since decided that such markup is largely overkill. The DOI uniquely identifies the publication already, and allows us to programmatically retrieve the rest of the data (title, authors, journal, etc) from semantically identified XML by querying against services such as CrossRef. This is the essential concept of linked data, by which both source and referer are enriched.
Moreover, DOIs follows a specific construction that lets us reliably identify them in plain text using regular expressions, making any futher semantics to declare that we are citing the article mostly irrelevant. This is convient for identifying all citations appearing in the notebook without any markup. The CITO example above has the advantage of providing a link and associating the DOI with the reason for the citation, by virtue of being inside the same html anchor element.
If we are not going to semantically mark up the reference list, we could consider abolishing the reference list all together. After all, as a tool for the digital reader the concept is rather vestigal – I hate losing my place by scrolling to the end of an article just to see to what reference number 7 refers. With the method shown thus far, the reader can open the link to access this information, but that still interrupts the flow of reading. The digitally native solution is a mouse-over or tooltip effect that displays this information, as many professional publishers already use in their HTML versions.
Once again, this is straight forward to support using the knitcitations package, at least for sites that include the popular bootstrap javascript libraries, such as this notebook. I have added an option to the in-text citation functions to provide such tooltips in a span element, such that calling the command
<span class='showtooltip' title='Shotton D (2010). "Cito, The Citation Typing Ontology." _Journal of
Biomedical Semantics_, *1*. ISSN 2041-1480, <URL:
http://dx.doi.org/10.1186/2041-1480-1-S1-S6>.'><a href='http://dx.doi.org/10.1186/2041-1480-1-S1-S6' property='http://purl.org/spar/cito/usesMethodIn' >Shotton (2010)</a></span>
This behavior can be toggled on by calling
cite_options(tooltip=TRUE)
after loading the knitcitations library. EDIT: Note that this requires the javascript trigger on the class showtooltip, which can be done by adding this to your header:
<script type="text/javascript">
$(document).ready(function (){
$(".showtooltip").tooltip();
});
</script>
Not all the literature we may wish to cite includes DOIs, such as arXiv preprints, Wikipedia pages, or other academic blogs. Even when a DOI is present it is not always trivial to locate. With version 0.4-0, knitcitations can produce citations given any URL using the Greycite API (Lord, 2012). For instance, this citation is created with the command citep("http://knowledgeblog.org/greycite", cito="usesMethodIn").
Github Issues Tracker: The Perfect Research Todo List [Lab Notebook - R] 2012-12-06 03:00
Github issues tracker has increasingly become my research to-do list. Far beyond bugs and features of the code associated with the project, the issues sign-post different directions for investigation and the progress I’ve made in each. Tags serve to group issues related to a common sub-project (as in my pdg-control) repo or priotize tasks (as in my nonparametric-bayes repo.
Issues not only have title and tags, but support a comment thread for progress and discussion of the issue. Thanks to github-flavored-markdown, issues can reference each other simply by number, and can be updated or closed automatically by mentioning the issue number in a commit.
Issues can also be grouped into shared deadlines, or milestones; a feature I haven’t fully exploited (but see our ews-review paper). In any collaborative project the issues can be assigned to different people, (though currently this requires they have a Github account).
A consequence of this workflow is a conveniently numbered, color-coded and cross-linked collection of steps involved in a given research project. This tends to be a higher-level overview than the individual commit log, particularly as I often use commits to track multiple runs with different parameters, or move code across to different supercomputers that do the actual runs.

pdg-controlI am still figuring out the right level or “resolution” on which to create and track issues.1 On one extreme, almost every commit could be seen as resolving an issue. The ideal use for me is probably nearer the other extreme, where individual issues are rather big-picture, and may be referenced by many commits. Perhaps the right way to think about it is that the questions addressed by resolving an issue are on the level of what is interesting to others, while changes in individual commits are more for me. Hopefully I get better at finding this relevant level.
Another nice feature of issues is that they can be closed when a particular line of investigation hits a dead-end, or stalls, or when the problem is resolved. Unlike the resulting paper from an investigation which essentially summarizes the issues that were closed successfully, the issues tracker also reveals the dead-ends, as well as those issues that were not closed a time of publication (but perhaps left to “further research”). Hopefully I will have some decent examples of this in the repositories accompanying my next papers.
Unlike issues, commits do not have a native tag structure (so-called “tags” mark important events in the commit history rather than grouping common commits). So at least this would group commits by the tag of the associated issue.↩
Citing Lab Notebook Entries [Lab Notebook - R] 2012-11-23 03:00
C. Titus Brown has an excellent post discussing his exploration into the merits and technicalities cross-posting his blog posts to figshare. The rfigshare package written by Ted Hart and myself can do just this, once we puzzled out some of the same challenges (Notes to Titus: though the documentation doesn’t mention it, you can get a programmatic list of available categories from http://api.figshare.com/v1/categories. Figshare can also take code as a fileset or dataset, and may soon add a type for it. The coolest thing about figshare is perhaps the way they add types in response to how they see users using the service).
The real discussion, though, is not about how, but why? Currently figshare doesn’t render the html or markdown/restructured-text source (other than as plain text), so it’s not a great place to read the posts. This may change in the near future as well, but the primary motivation for doing this seems to be on the ability to get an honest-to-goodness DOI for your entry. So why a DOI?
The answer most often put forward is that “a DOI facilitates citing a paper in the formal literature.” I think that’s not entirely accurate. For one, the formal literature has no trouble citing things that do not have a DOI. Perhaps the answer is meant to mean track citations to my content, e.g. for statistical/impact purposes. This may be closer to the mark, but it still needs work. What counts depends on whose doing the counting. Anyone browsing the citation counts of a work through different mediums has surely encountered Thompson-Reuters has very specific criteria to be included, so I don’t believe they are tracking citations to anything with a DOI. Nor, I think is Scopus. Perhaps the target is Google then?
If you want Google Scholar to count the number of citations to your blog post, I have good news for you. You do not need a DOI and cross-posting to figshare. You just need to follow the metadata requirements as outlined by Google Scholar itself. This will help Google Scholar identify your blog post as a academic object, and add it to your profile. Anything indexed by Google scholar that cites your blog post will count as a reference. The basics are really simple. Have metadata such as:
<meta name="resource_type" content="Lab Notebook"/>
<meta name="citation_journal_title" content="Lab Notebook"/>
<meta name="citation_publication_date" content="2012-11-23 00:00:00 -0800"/>
<meta name="citation_date" content="2012-11-23 00:00:00 -0800"/>
<meta name="citation_author" content="Carl Boettiger"/>
<meta name="citation_title" content="Citing Lab Notebook Entries"/>
Just replace Lab Notebook etc. with the correct values for your site. The example above will do so automatically if you use Jekyll using information specified in the YAML header. It seems the entry also needs a section called either “References” or “Bibliography” followed by a list of references. Not sure what format Google Scholar will do best at disambiguating. (Note that what I refer to as “Google Scholar” metadata looks like it was originally the convention adopted by HireWire Press.)
Phil Lord and colleagues have written the fantastic API called greycite [Lord 2012], which can be used to generate citation data from any website that has semantic markup (including HTML5 semantics, also Dublin Core or OpenGraph ontologies and Google Scholar metadata). The API can return citations formatted in bibtex or JSON. Try it out at greycite.knowledgeblog.org
Now there is another reason to use DOIs and figshare, which is, I think, entirely independent – archival preservation (decoupled journal, anyone?). DOIs are potentially more permanent than URLs (though I’ll leave that debate to the experts). Figshare content is backed up by CLOCKSS. Forever is a long time, but this certainly sounds like a better archiving strategy. Figshare should be providing the necessary Google Scholar metadata on each object now. My current practice has been to archive my notebook in annual chunks on figshare (posting entries individually feels fragmented and cluttered to me), and rely on the more native web solution of HTML metadata to allow my entries to “citable”. Perhaps this is not ideal, but for the short term, the content is discoverable and citable via Google Scholar, which points to my address. If my site vanishes from the web, one might hope that an academic search for my lab notebook might recover the content from the archived version.
I stumbled across Google’s indexing of some of my lab notebook entries somewhat by accident. For instance, one of my entries on optimal control in which I had listed some references started turning up in Google Scholar Being able to engage in a scholarly exchange through blogs that can cite and be cited by the formal literature certainly sounds like an important step forward in generating new publishing models. Of course it is also obvious that the procedure I have outlined could be trivially exploited for some rather blatant gaming of Google Scholar’s citation statistics. This post certainly is not an endorsement of gaming such statistics. If nothing else, perhaps this once again underscores the weakness of reliance on metrics of quantity when trying to infer quality. Will we ever let go of that fallacy?
Semantic Lab Notebook [Lab Notebook - R] 2012-10-14 03:00
As the lab notebook grows, to make the maximum use of content it would be particularly useful to maximize the ability for a computer to understand the content, allowing us to identify, manipulate, and connect data using scripts and software. This is the concept of linked data, or a semantic notebook. I have explored this this idea before in the context of a wordpress-based platform, but now that Jekyll has let me strip away some of the abstraction of Wordpress it seems a good time to revisit this idea.
Already the notebook is written in HTML5, which has considerable semantic structure compared to it’s predecessors. HTML5 introduces the structural elements
<nav><header><article><section><aside><footer>that intuitively define the organization of an page, setting off the important content from the window-dressing. HTML5 also defines inline elements <time> (see the links in Caveats), and <mark>, and the existing tags for metadata, which let us specify <title>, and basic <meta> tags for metadata such as author, <meta name="author" content="Author Name">, encoding, description, and keywords. Links <link> and <a> use the rel attribute to describe the link target. Though one can write anything at all in this text, HTML5 defines a small vocabulary with recognized meaning, some old, some new, such as <a rel="tag", rel="license" as well as the older <link rel="stylesheet" href="style.css"> and <a rel="nofollow". Here is a great overview of the semantics in HTML5, with a bit more about the available . To expand our vocabulary beyond these elements, however, we will need more tools.
W3C confusingly provides two standards for formally defining semantic content using an external vocabulary or ontology. The first is microdata, introduced as a simpler alternative to the second, RDFa, an HTML-adaptation of the RDF XML standard (originally developed for the now-defunct XHTML 2.0).
Microdata introduces new attributes into HTML tags like <div>, <span>, <h1>. The first of these is itemtype, which points to an external resource such as schema.org to define the vocabulary. To have this vocabulary apply to child elements, we just add the attribute itemscope. Then we can set the value of attribute itemprop in this or following elements to give it semantically defined meaning, such as
<div itemscope itemtype="http://data-vocabulary.org/Person">
<h1 itemprop="name">Carl Boettiger</h1>
<a itemprop="url" href="http://www.carlboettiger.info">http://carlboettiger.info</a>
<img itemprop="photo" src="https://en.gravatar.com/userimage/12904315/7edea703b826fbbe07f2ae4d95b8416b.jpg?16"/>
</div>
where the terms such as name and url have precise meanings attached to “Person”, as specified at the “http://data-vocabulary.org/Address”. This ability to point to an external vocabulary is really the key concept of linked data. In the spirit of HTML5, microdata is much simpler than RDFa, but also more limited. Here is an excellent comparison, but for our purposes we will use RDFa as it is more common in academic use and will more seamlessly allow us to use academic ontologies. (While microdata has a clear mapping to RDF, it is not clear that any ontology that can be expressed in RDF can also be expressed in microdata).
RDFa can be written in a very similar manner. The itemprop attribute is replaced by the property attribute, and vocab replaces itemtype and automatically implies itemscope to child nodes.
<div vocab="http://xmlns.com/foaf/0.1/" typeof="Person">
<h1 property="name">Carl Boettiger</h1>
<a property="homepage" href="http://www.carlboettiger.info">http://carlboettiger.info</a>
<img property="depiction" src="https://en.gravatar.com/userimage/12904315/7edea703b826fbbe07f2ae4d95b8416b.jpg?16"/>
</div>
We could have used only the property attribute along with the complete URI to the term definition instead of this structure. typeof indicates that the child elements are all of the same type (in this case, all belong to the same “Person”). If we were omitting vocab and writing out all URIs, we would have had <div typeof="http://xmlns.com/foaf/0.1/Person"> as well. In RDFa, we also have the resource attribute, which allows us to specify a URI for the element being described. This could be a relative or absolute URL. If the object is a link, using resource is not necessary, and the property is taken to describe the URL given in href or src rather than the anchor text.
Two older options lie at either extreme: the most basic is the older technique of microformatting, basically relying on standard class and rel attributes to convey semantic information. Simple and without new attributes, but probably too limited for our purposes so we won’t concern ourselves with it further. The other venerable approach is serve the page as XHTML, which renders in the browser in much the same way but can be parsed by a machine as XML, with all the power and extensibility that presents. Unlike earlier HTML standards, HTML5 is already valid XML (already “serialized”), so the same page can be served in either format (earlier specs were SGML, standardized general markup languages, of which XML is just a subset). To allow parsing of the HTML5 pages as either type (see polyglot), we need only add the language and namespace to the opening <html> tag,
<html lang="en" xmlns="http://www.w3.org/1999/xhtml" xml:lang="en">
An essential caveat is that these are all new approaches which may not render well in legacy browsers, particularly Windows Internet Explorer. Some of these, like recognizing and styling the HTML5 semantic elements in IE, can be addressed in CSS, for which Twitter Bootstrap does a decent job. Another caveat The HTML5 spec has not been finalized, and some things are still in flux, as the removal and reinstatement of the <time> element illustrates.
Now that we’ve familiarized ourselves with the options, it’s time to see what semantic content we can implement.
| Content/Data | Example types | Links to Potential vocabularies |
|---|---|---|
| Page structure | <header> |
HTML5 |
| Post metadata | keywords, timestamps | Schema.org microdata or Dublin Core |
| Author metadata | Name, contact, networks, | FOAF, Dublin Core |
| interests, publications) | ||
| Licenses | CC0 | CreativeCommons |
| Citations | bib info, reason for citing | CiTO, BiRO, |
| bibo, Dublin Core | ||
| Taxonomic data | species names | Darwin Core |
| Ecological data | measurements, units, etc |
In another entry I will try and highlight where and what semantic content I have added (work in progress), with examples of each vocabulary. The first four types are relatively static content that can be easily woven into the Jekyll template files. Using Jekyll & Liquid to pull in template information from the _config.yaml should help avoid repetitive entry and make updating the linked data easier. The last three are entry-specific content, and will be more challenging. I hope to add semantic support to knitcitations, including the option for CiTO types, which should make entry of citation data quick and easy (SO question illustrating semantic citation).
The last two are much richer, specific vocabularies. For the moment, it might be best to use these to give more precise meaning to tags, which are already used as metadata on posts. This would allow posts to be still created in simple markdown without the burden of adding in lines of RDFa and cluttering the markup. Meanwhile full datasets provided in EML are likely to live as separate files, rather than as a random table in the middle of a notebook entry.
As always, feedback, corrections or suggestions are appreciated!
Welcome to my Lab Notebook - Reloaded [Lab Notebook - R] 2012-09-28 03:00
Welcome to my lab notebook, version 3.0. My original open lab notebooks began on the wiki platform OpenWetWare, moved to a personally hosted Wordpress platform, and now run on a Jekyll-powered platform (site-config), but the basic idea remains the same. For completeness, earlier entries from both platforms have been migrated here. Quoting from my original introduction to the Wordpress notebook:
Disclaimer: Not a Blog
Welcome to my open lab notebook. This is the active, permanent record of my scientific research, standing in place of the traditional paper bound lab notebook. The notebook is primarily a tool for me to do science, not communicate it. I write my entries with the hope that they are intelligible to my future self; and maybe my collaborators and experts in my field. Only the occasional entry will be written for a more general audience. […] In these pages you will find not only thoughts and ideas, but references to the literature I read, the codes or manuscripts I write, derivations I scribble and graphs I create and mistakes I make.
My original introduction to the notebook from November 2010 dodged this question by suggesting the exercise was merely an experiment to see if any of the purported benefits or supposed risks were well-founded. Nearly three years in, can I draw any conclusions from this open notebook experiment?
In that time, the notebook has seen six projects go from conception to publication, and a seventh founder on a null result (see #tribolium). Several more projects continue to unfold. I have often worked on several projects simultaneously, and some projects branch off while others merge, making it difficult to capture all the posts associated with a single paper into a single tag or category. Of course not all ideas make it into the paper, but they remain captured in the notebook. I often return to my earlier posts for my own reference, and frequently pass links to particular entries to collaborators or other colleagues. On occasion I have pointed reviewers of my papers to certain entries discussing why we did y instead of x, and so forth. Both close colleagues and researchers I’ve never met have emailed me to follow up on something they had read in my notebook. This evidence suggests that the practice of open notebook science can faciliate both the performance and dissemination of research while remaining compatible and even synergistic with academic publishing.
I am both proud and nervous to know of a half dozen other researchers who have credited me for inspiring them to adopt open or partially open lab notebooks online. I am particularly grateful for the examples, interactions, and ideas from established practitioners of open notebook science in other fields. My collaborators have been largely been somewhere between favorable and agnostic towards the idea, with the occasional request for delayed or off-line notes. More often gaps arise from my own lapses in writing (or at least being intelligible), though the automated records from Github in particular, as well as Flickr (image log), Mendeley (reading log), and Twitter and the like help make up for some of the gaps.
In creating my wordpress lab notebook, I put forward the idea of an “Integrated Lab Notebook”, a somewhat convoluted scheme in which I would describe my ideas and analyses in Wordpress posts, embed figures from Flickr, and link them to code on Github. Knitr simplified all that. I can now write code, analysis, figures, equations, citations, etc, into a single Rmarkdown format and track it’s evolution through git version control. The knitr markdown format goes smoothly on Github, the lab notebook, and even into generating pdf or word documents for publication, never seperating the code from the results. For details, see “writing reproducibly in the open with knitr.”
You can page through the notebook chronologically just like any paper notebook using the “Next” and “Previous” buttons on the sidebar. The notebook also leverages all of the standard features of a blog:
I use categories as the electronic equivalent of separate paper notebooks, dividing out my ecological research projects, evolutionary research topics, my teaching notebook, and a few others. As such, each entry is (usually) made into exactly one category. I use tags for more flexible topics, usually refecting particular projects or methods, and entries can have zero or multiple tags.
It can be difficult to get the big picture of a project by merely flipping through entries. The chronological flow of a notebook is a poor fit to the very nonlinear nature of research. Reproducing particular results frequently requires additional information (also data and software) that are not part of the daily entries. Github repositories have been the perfect answer to these challenges.
My Github repositories offer a kind of inverted version of the lab notebook, grouped by project (tag) rather than chronology. Each of my research projects is now is given it’s own public Github repository. I work primarily in R because it is widely used by ecologists and statisicians, and has a strong emphasis on reproducible research. The “R package” structure turns out to be brilliantly designed for research projects, which specifies particular files for essential metadata (title, description, authors, software dependencies, etc), data, documentation, and source code (see my workflow for details). Rather than have each analysis described in full in my notebook, they live as seperate knitr markdown files in the inst/examples directory of the R package, where their history can be browsed on Github, complete with their commit logs. Long or frequently used blocks of code are written into functions with proper documentation in the package source-code directory /R, keeping the analysis files cleaner and consistent.
The issues tracker connected to each Github repository provides a rich TO DO list for the project. Progress on any issue often takes the form of subsequent commits of a particular analysis file, and that commit log can automatically be appended to the issue.
When scripting analyses or writing papers, pretty much everything can be captured on Github. I have recently added a short script to Jekyll which will pull the relevant commit logs into that day’s post automatically. Other activities fit less neatly into this mold (reading, math, notes from seminars and conferences), so these things get traditional notebook entries. I’m exploring automated integration for other activities, such as pulling my current reading from Mendeley or my recent discussions from Twitter into the notebook as well. For now, feed for each of these appear at the top of my notebook homepage, with links to the associated sites.
Migrating From Wordpress To Jekyll [Lab Notebook - R] 2012-09-19 03:00
Thanks to a recent bugfix in the exitwp scripts I was able to export all my Wordpress entries. The script pulls the entries from the Wordpress database and formats them in markdown, along with extracting all metadata such as timestamp, tags, categories, publication status, and Wordpress id number, which are all embedded as YAML header information.
When I moved to the Jekyll notebook, I left the Wordpress site standing with all its existing content and simply remapped the home pages. While it was nice to have the Wordpress system as a fall back while I got used to Jekyll, it meant that I couldn’t benefit from the faster and lighter nature of Jekyll while much of my traffic occurred on the Wordpress pages. I was running a dedicated virtual private server for the Wordpress hosting, and still the site wasn’t very responsive. To get an idea, try “flipping through the pages” of the new Jekyll notebook using the “next” and “previous” post buttons. The new pages load immediately, making it easy to flip through looking for some word or image I can’t find with tags or searches. Flipping through pages is the time-honored way of interacting with paper notebooks, so I’m glad that the new system is responsive enough to imitate this well.
Migrating files using exitwp is a relatively seamless affair. Unfortunately, my Wordpress posts make rather heavy use of plugins, which work through shortcodes that still appear in the converted files, such as [flickr], [latex], [code lang="r"], and [cite], for image embedding, equation embedding, syntax highlighting, and citations by DOI, respectively. While a suite of Jekyll plugins exist for each of these tasks, I’d like my regular posts to be pure markdown (other than the YAML header), making it easy to migrate or reuse the content in the future in different platforms. Here is the R script I used for converting my shortcodes, as described below.
For instance, several markdown extension languages including, Github-flavored markdown, already allow for syntax-highlighting with fenced code blocks. I wrote a short script to replace all (most) of the shortcodes in use on my Wordpress site. (Over the years, I’d actually used a variety of different syntax highlighting plugins, making it necessary to match a variety of different shortcodes). Other than matching a lot of patterns, this was pretty straight forward.
My workflow pushes all the images and graphs I create to flickr when the script runs. (I could also automatically push the images to Wordpress, or simply to my own site, but started using flickr pretty early on for this. The SOAP API is excellent, as is the Rflickr R package) Images were embedded into my Wordpress site using the shortcode and image ID number. To generate stand-alone markdown, I wanted tp replace all the image codes with the flickr URLs to the images, which is a trivial task with the API and Rflickr.
Again I had used a variety of shortcodes for latex plugins before discovering MathJax, so I had to handle a variety of syntaxes here. Previously I’ve tried to write equations in a manner compatible with the redcarpet markdown parser that powers Github-Flavored Markdown (see the corresponding Jekyll plugin), e.g. surrounding equations with <div> blocks to avoid the parser mangling them. Instead, I’ve found it easier now to switch to pandoc for my markdown parser (and corresponding Jekyll Plugin). Pandoc uses the basic TeX $ and $$ for inline and display equations, rendering it into the MathJax (and modern LaTeX standard) \( and \[ on conversion. Being pandoc-compatible is quite nice, making it easy to create an e-book, epub, or latex-based pdf of my notebook. I hate having to use a markdown syntax that isn’t compatible with most other extended markdown parsers, but with so many different, inconsistent parsers this cannot be easily avoided. Regardless, replacing the various shortcodes to the Pandoc syntax is just a simple regexpr step.
Many of my posts relied on kcite to generate bibliographies for the post using DOIs. A short call to my knitcitations package allowed me to generate the parenthetical citations and bibliographies instead. In principle this step could be run at the time I compile the site with Jekyll. Pages would be valid .Rmd, needing knitr as “markdown” interpreter. This is an intriguing way to go, but probably too complicated for the moment. Running additional R code would be nice, but could slow the time needed to build the site, even with knitr’s caching. Meanwhile, converted pages simply have the citations already processed and the bibliography added in as HTML at the end of the post.
My Jekyll site uses the SEO recommended structure of year/month/day/page-title for URLs, while my Wordpress site used simple random id numbers. To ensure that links to the old Wordpress pages resolve to the newly migrated pages, I added a little Jekyll plugin, redirects.rb. One of the elegant things about Jekyll is the ability to create such plugins that do this just the way you would image doing so – creating auxiliary pages at each of the redirect URLs with a simple redirect command. To install, place redirects.rb in _plugins, redirects.html in _layouts, and in _config.yml add the line redirects: yes (see site source).
I am using Disqus as the comment engine for entries. Since URLs change in the above step and I do not provide a unique disqus code to each page, I am attempting to migrate comments using the Disqus “migrate threads” tool, which simply takes a csv file listing old and new urls in consecutive columns. Will have to wait and see if this works.
Several entries on the Wordpress site were marked private, and only visible after administrative login. Most of these entries are actually solicited journal reviews of various manuscripts which I cannot make public. exitwp marks these posts as published: false in the metadata, so it is easy to remove them from the Jekyll source site. They can be managed in a separate Github-excluded directory (so that they cannot be seen on the website source pages on Github either) that can be .htaccess password protected.
Categories and tags are handled automatically in the conversion. One of the greatest things about having all the content on a common system is the ability to share a common tag-pool.
If you follow the notebook through RSS feeds, all the migrated posts will be showing up as new unread entries, as the atom feed dates entries by when they appear, not the stated post date. My apologies!
A couple posts have rare shortcodes which are probably easier to correct by hand. My apologies for any rough edges resulting from the conversion (or the exchange of markdown parsers). Hopefully I’ll get these ironed out in the near future, but feel free to leave a comment on the page or the issues tracker on any such errors.
Esa Changes Arxiv Policy Following Community Comments [Lab Notebook - R] 2012-09-05 03:00
Earlier today, Scott Collins, the president of the Ecological Society of America has announced that the society will now accept articles that have previously been posted on preprint servers. This comes on the heels of a growing discussion in our community. Ethan White has a good summary over on Jabberwocky Ecology.
Many voices have joined the discussion over the past month, and it is exciting and vindicating to see the Society engage and discuss these questions. With this announcement out, I thought I might share my original letter. Here’s the text of an email I sent on August 1st to Don Strong, editor-in-chief of Ecology and a friend to open science known for his bottom-up view that ecologists should show their publication preferences by their actions. Don kindly forwarded this email to other members of the board.
Dear Don,
Perhaps you have already seen the perspective appearing in Nature this week, “Geneticists eye the potential of Arxiv.” I must say it is particularly saddening to see the Ecological Society of America being singled out in the discussion as a professional society that opposes pre-prints, particularly given that ESA is not beholden to corporate publishers, and in the face a rising swell of interest in pre-print server capacity, as evidenced by the Nature article (which quotes our own Graham Coop), the emergence of an NCEAS working group to bring about a preprint server, and the extensive online discussions about ESA’s stance against pre-prints which was no doubt responsible for bringing ESA into the negative limelight of this perspective.
The discussion launched by Professor Ethan White on his blog suggests that ESA has in fact only recently removed a clause in the policies explicitly permitting the use of the arXiv. Meanwhile members of the NCEAS working group had considered approaching ESA to partner in their efforts to bring about a preprint server and culture, hoping to take advantage of the agility ESA has as a respected and independent society publisher. Ecology as a field has been a leading example of pushing for innovative and open publishing practices such as mandatory data archiving, and ESA has led this front for decades with innovations such as Ecological Archives and more recent Ecosphere. It shames me to see our society and our field painted as a backwater of regressive policies in such prominent magazines when even journals such as Nature, Science, & PNAS permit and encourage the use of preprint servers such as the arXiv. I would hate to see our most innovative research migrate away from the Society at a time when ESA could be leading our field through this academic publishing transition that is now discussed broadly in the NY Times, Guardian, Economist, & USA Today. This is a chance for the Ecological Society to both lead and flourish.
I hope that these issues can be discussed in some fashion with the input of our community at the annual meeting in Portland. The British Ecological Society already plans to use the meeting to discuss a digital future of their journals, surely ESA will be doing as much at it’s own meeting, formally or informally?
Thank you for your time and consideration. Please let me know if there is anything I can do to help.
Cheers,
Carl
knitr, github, and a new phase for the lab notebook [Lab Notebook - R] 2012-03-21 08:13
I have recently modified the basic workflow of my lab notebook since discovering knitr. Before, I would write code files which I could track on github, push figures created by the code to flickr, and then write a notebook entry on wordpress describing what I was doing. I’d embed each figure I wanted into the entry, and each figure got an automatic link to github for the script that created it (which usually worked, though it didn’t say where in the script the command came from, and it required manually specifying the script name).
Because knitr allows me to write a single file containing code and formatted text, and will automatically display the code and embed the images, I can avoid that more convoluted workflow and just write.
](https://github.com/cboettig/pdg_control/blob/master/inst/examples)
What makes this so excellent is that knitr allows me to write in markdown, and github automatically displays nicely formatted markdown instead of raw script when you visit the page. So whereas before I would keep a bunch of working R scripts in projectname/demo I now keep a bunch of markdown scripts in inst/examples, ((since I’m using the R package for projects and demo/ doesn’t want non-R scripts)).
The great thing about this is that I can just click on each script and see nicely rendered text, links, code, and figures right on github.
](https://github.com/cboettig/pdg_control/blob/master/inst/examples/Reed.md)
While I can push this same markdown script to wordpress and have it be rendered in my notebook, I think maintaining these examples on github is preferable. Note that every script-name appears twice, once with and once without the _knit_ extension. The _knit_ extension indicates the file I ran to create the output (the code is in html comments so you can only see them in raw form). Because all the code is displayed in the output file (unless I call knitr options to surpress this), there’s really no need to view the _knit_ file to reproduce the example, everything is in one place in the output .md file.
These files benefit from the github managed version history, just clicking the history button gives a list of all the former versions, with code and results right there.
While I could update a post in the notebook in the same way, the version control of this wordpress notebook is more crude, and more importantly, the blog-format is designed for a linear flow, whereas in a given day I might update each of these example scripts. This seems like a much more natural workflow then having consecutive entries in the notebook with updated versions of the analysis the day before, and more natural than going back and changing a previous notebook entry.
](https://github.com/cboettig/pdg_control/commits/master/inst/examples/model_uncertainty.md)
Github can also gives nice snapshot of what’s changed, along with the commit notes for each project, for each day. Now that I can display figures and formatted text on github, as well as code, what role does the Wordpress notebook play? I think this wordpress notebook can resume is proper role as a lab notebook, containing reflections and synthesis on what I’ve done, rather than the more comprehensive copies of each analysis and each figure. Because it’s the internet, I can link to each of the analyses of that day using version-stable links from github, or links that always give the most recent version. This requires additional effort, but it’s a reflection I should be doing anyway. We’ll see how it goes. Meanwhile, welcome to the open lab notebook v2.0.
When I write code longer than a few lines, I try to make it a function, or collection of functions, and include basic Roxygen-style documentation with it so I don’t have to read the code to remember how to use it. These functions naturally live in the R/ directory of the project. The project’s R package can be installed, and all these functions are then available. Each of my example scripts calls functions belonging to the package, but those functions change less regularly. To fully reproduce the example, it would be necessary to grab a copy of the R package from the same commit-version as the script. In practice, most of the time any version of the package R functions could be used.
Github has wiki pages which I could use instead of putting my entries in inst/example, since both render markdown (the wiki will additionally render mathjax math, all be it as a png). However, the wiki is aimed at online editing, and exists as a separate repository, so just keeping the markdown files in the project directory is simpler.
Github doesn’t actually host the images. In fact, my images are still being pushed to flickr, and you can see them there. Knitr is handling this automatically as the figures are created, keeping track of the links so they can be included in the output markdown. Knitr can do this with imgur out of the box, and I’ve also written a wrapper to let it push the images to a wordpress site. Doesn’t really matter where they are stored, they can always be viewed on Github.
I’ve found that the knitted markdown examples make the most sense for fast-running examples. Despite excellent caching support, I’ve found it best to run really long-running examples as external R scripts, and then save and import this data. Such scripts are often being run on a cluster that can’t push images to the internet anyway.
It occurs to me that this system would be harder but not impossible, in a closed environment. I could upload the figures to flickr with private status. The links are hashes, so cannot be guessed. If the output were then hosted on a secure site (running a markdown renderer, such as Jekyll), instead of github, these links would still work to display the images. Then one could give selected access to those pages. But the open solution works out of the box.
Citing R packages [Lab Notebook - R] 2012-03-20 06:02
I’m not always careful in citing all the R packages I use. R actually has some rather nice built-in mechanisms to support this, so I really have no excuse. Here’s some quick examples:
citation("ouch")
To cite the ouch package in publications
use:
Aaron A. King and Marguerite A. Butler
(2009), ouch: Ornstein-Uhlenbeck models
for phylogenetic comparative hypotheses (R
package),
http://ouch.r-forge.r-project.org
Butler, M. A. and King, A. A. (2004)
Phylogenetic comparative analysis: a
modeling approach for adaptive evolution
Am. Nat. 164:683--695
As ouch is continually evolving, you may
want to cite its version number. Find it
with 'help(package=ouch)'.
Can I have that in bibtex format please?
toBibtex(citation("ouch"))
@Manual{,
title = {ouch: Ornstein-Uhlenbeck models for phylogenetic comparative hypotheses},
author = {Aaron A. King and Marguerite A. Butler},
year = {2009},
url = {http://ouch.r-forge.r-project.org},
}
@Article{,
author = {Marguerite A. Butler and Aaron A. King},
title = {Phylogenetic comparative analysis: a modeling approach for adaptive evolution},
journal = {American Naturalist},
year = {2004},
volume = {164},
pages = {683--695},
url = {http://www.journals.uchicago.edu/AN/journal/issues/v164n6/40201/40201.html},
}
Notice that this package provides the citation information for both the package and the associated journal article simultaneously, and R has successfully identified the formats as ‘Manual’ and ‘Article’ respectively.
After running your code, consider creating a custom bibtex file containing the citation information for all the packages you have just used. (The file can be imported into most citation managers, if LaTeX isn’t your thing).
sink("test.bib")
out <- sapply(names(sessionInfo()$otherPkgs),
function(x) print(citation(x), style = "Bibtex"))
You can also simply generate the list of loaded package in LaTeX format, which could be automatically included.
toLatex(sessionInfo(), locale = FALSE)
R will attempt to automatically construct the citation information for the package automatically from the description file, so it is not strictly necessary to do anything to your package to create it. Note that R has recently adopted a new syntax to specify the authors, which is a bit more precise. Instead of using Authors: in the DESCRPTION, we use:
Authors@R: c(person("Carl", "Boettiger", role = c("aut", "cre"), email = "cboettig@gmail.com"),
person("Duncan", "Temple Lang", role = "aut"))
This defines the roles (author, creator, etc, see ?person for details), and ‘creator’ takes the place of the Maintainer: designation, and requires an email address. If you wish to add an additional publication as part of the citation information (such as the example from ouch above, you can specify this in the CITATION file. For the example this looks like:
citHeader("To cite the ouch package in publications use:")
citEntry(entry = "Article",
author = personList(as.person("Marguerite A. Butler"),
as.person("Aaron A. King")), title = "Phylogenetic comparative analysis: a modeling approach for adaptive evolution",
journal = "American Naturalist", year = 2004,
volume = 164, pages = "683--695",
url = "http://www.journals.uchicago.edu/AN/journal/issues/v164n6/40201/40201.html",
textVersion = paste("Butler, M. A. and King, A. A. (2004)",
"Phylogenetic comparative analysis: a modeling approach for adaptive evolution",
"Am. Nat. 164:683--695"))
citFooter("As ouch is continually evolving, you may want to cite its version number. Find it with 'help(package=ouch)'.")
[1] "As ouch is continually evolving, you may want to cite its version number. Find it with 'help(package=ouch)'."
attr(,"class")
[1] "citationFooter"
(It seems like there should be a simple way to generate this automatically from the bibtex format, but I haven’t discovered it.)
I wrote an R function for the Crossref API in our rplos package. We should probably be formatting the output as an R bibentry, taking advantage of R’s understanding of citation structure. Then I could work automatic citation look-up into my posts using inline knitr calls, such as:
print(crossref("10.1038/44766"), style="text")
An alternate mechanism could read in a local bibtex file and drop in the correct entry in the desired format.
Journey to freedom - a code's tale of open source license migration [Lab Notebook - R] 2012-01-31 12:58
My software wants to be free. It wants to be seen and used and loved by as many people as possible. When first it heard of open source licenses, it set sail to join the company of great software in the promised land, but finding true freedom has been a tortured journey.
Created and defended by the Free Software Foundation and used by such venerable institutions as the Linux Kernel, the gcc compiler, and the R statistical environment, the GNU’s General Public License seemed like a gold standard to call home. Many of my codes quickly migrated to this beacon of light, while others, already making use of other GPL licensed software, seemed compelled to follow by it’s viral share-alike clauses. But my code wasn’t free. It couldn’t play nicely with users from the pantheon of other open source licenses, upsetting free software developers and university tech transfer offices alike. It was scorned by open science advocates and repositories. And when it wanted to leave and migrate to a more free and permissive license, it felt trapped.
By now the software was written as an R package (combining C source code and R functions), depended on several R packages, and even compiled against the GSL C library, all GPL licensed software. Surely it was stuck?
Only after close examination did the package realize that it could escape. It’s not a derivative work. It’s not distributing the other R packages or R itself. That software have to be installed separately – it is only dynamically linked ((called at runtime, not compiled into the binary libraries of my package)) to my package. The case is less obvious for the dependency of the C library on the GSL code, since this must be compiled to run. If the C functions provided by the GSL are compiled statically, they are pulled from their source and crammed into the binary next to my own code – making a derivative work that doesn’t need the GSL libraries installed separately to run. Fortunately that is not the case, these functions are also dynamically linked, and my code is free to migrate.
Fleeing the convoluted and tortured shore of GPL, my code happily reached the land of BSD. In it’s forth iteration as FreeBSD, with only two simple clauses, it seemed anything was possible. The only thing it does require are that the license always be distributed with the source code or binaries of the software. This makes it difficult to apply to repositories that also archive data – facts about the world owned under anyone’s copyright – and other non-code material such as documentation that may be found in an R package or an academic data repository. For this reason, the Dryad repository objects to that little clause of redistributing the FreeBSD license, and insists on public domain declaration provided by the CC0 license.
Ah, so we arrive at the public domain, or as close as I can legally get. While this seems to be the ultimate freedom my code has long sought, it finds itself rather lonely on these foreign shores. The Open Source Initiative doesn’t yet recognize it, which means it doesn’t meet the “open source license” requirements of academic journals like PLoS Computational Biology or academic conferences like Bioinformatics Open Source Conference or iEvoBio. It has not yet been used for R packages on the CRAN repository (see below), and would require using R’s custom license file mechanism rather than a simple LICENSE: CC0 line in the package meta-data. It does get a mention for being GPL compatible by the FSF ((i.e. you can redistribute it under the GPL)), but the license that isn’t a license needs more support. Following their guidelines, I’ve written the Open Software Initiative hoping it will gain that recognition.
Hello,
I would like to submit the Creative Commons zero license for consideration. I am new to this list, forgive me if it has already been considered. I understand that this license has been considered GPL compatible by the FSF with this recommended format.
Submission type: Approval License Name: Creative Commons Zero, CC0 Category: Licenses that are popular and widely used or with strong communities The legal code as plain text.
The scientific community is increasingly embracing this option as the most open and compatible license. It is required by scientific data repositories (that also archive scientific software, with cite-able DOIs) such as Dryad.
I feel it is important that this license achieve recognition by the Open Source initiative, as certain scientific journals & conferences permit only OSI recognized licenses, (e.g. PLoS Computational Biology requirements or Bioinformatics open source conference requirements).
R uses a simple but powerful license format in the DESCRIPTION files of all packages, which contain the essential meta-data for the package. Common FOSS licenses are recognized automatically (including version numbers or version inequalities). Since CC0 doesn’t (yet) make this list, we can use a custom license file where we provide the full plaintext CC0 license in the file LICENSE and write “file LICENSE” in the DESCRIPTION (my example).
We can also see the frequency with which various licenses are used on the R repository, CRAN, with a few lines of code:
p = available.packages(contrib.
url("http://cran.r-project.org"), type = "source")
sort(table(p[, "License"]))
Are open lab notebooks considered prior publication? [Lab Notebook - R] 2012-01-16 05:47
This question invariably comes up at some point in any discussion of open notebook science. This concern is usually voiced in reference to the high-visibility magazines, which many scientists seem to assume will have very restrictive conditions. A quick read of their policies shows otherwise. Here are the links to pre-publication policies of major journals, with my short summaries & comments.
Nature - Go right ahead. Explicit protection clause for open/collaborative blogs/wikis. Just don’t talk to the press – guess that could include tweeting. Also Nature would like to see those links when you submit.
PNAS - Nope, not prior publication here either. In fact, PNAS has one of the most liberal stances on prepublication which is well worth reading. Even summaries of the work in the media encouraged – they only remind authors not to forget to publish!
Science - The policy listed there sounds more conservative (phrases like “most cases” and “contact the editors” don’t inspire confidence). Luckily, actually contacting the editors does: Deputy editor Brooks Hanson writes to me:
We allow posting on not-for-profit pre-print archives, so posting on arxiv.org is fine at any time. we ask that if you post before acceptance that you do not indicate that the paper is submitted to Science.
As to lab notebooks, it would probably depend on what was in the “notebook” but if it were just notes or thoughts, it would probably not be a great concern.
Many discipline-specific journals don’t state their policy on this as clearly in their advise to authors. Of course writing to the editors is the best way to get clarification, but I think their are reasonable proxies to guess what’s okay and what’s not.
Posting on preprint servers in advance of submission is probably a greater threat to the journal’s exclusive control of the content than the less-digestible and less accessible (decentralized) posting of an open notebook, and as it’s a more widespread practice it may be a good signal to look for. F1000 also compiled a list of journals that would consider archiving a poster as prepublication, which could be used as another proxy – if they are okay with posters, notebooks are probably in the clear. The Sherpa/Romeo project provides links to the pre-publication policies of many journals. Its database also identifies which journals allow what level of public archiving. Search for your favorite journals there. To the extent that “Yellow” refers “at the time of submission,” rather than post-publication, one might assume that publisher wouldn’t much object to an open notebook. Of course, I’d love clarification.
RoMEO Colour
Archiving policy
Can archive pre-print and post-print or publisher’s version/PDF
Can archive post-print (ie final draft post-refereeing) or publisher’s version/PDF
Can archive pre-print (ie pre-refereeing)
Archiving not formally supported
For instance, looks like the Royal Society journals are okay too. They receive a Green status, permits pre-review author’s copy on preprint servers. Published copy uses Creative Commons Attribution (cc-by) licensing. Their duplicate publishing statement makes no reference to archives, only other publishers.
How google views me: by search terms or citation counts? [Lab Notebook - R] 2011-09-09 05:55
How Google search views me: here’s a word cloud of search terms reaching my site in August. Word cloud produced with R: click-through for link to source-code. Uses the rather convenient tm package for text-mining functions in R. Note that this shows the frequency of individual words used in searches, rather than the whole search term.
Code:
require(tm)
require(wordcloud)
require(RColorBrewer)
carl <- read.csv("searchterms.csv", colClasses=c("character", "numeric"))
words <- character(sum(carl[[2]]))
m <- 1
for(i in 1:length(carl[[1]])){
n <- carl[[2]][i]
x <- carl[[1]][i]
words[m:(m+(n-1))] <- rep(x, n)
m <- m+n
}
carl <- Corpus(DataframeSource(carl))
carl <- tm_map(carl, removePunctuation)
carl <- tm_map(carl, tolower)
carl.tdm <- TermDocumentMatrix(carl)
carl.m <- as.matrix(carl.tdm)
carl.v <- sort(rowSums(carl.m), decreasing=TRUE)
carl.d <- data.frame(word=names(carl.v), freq=carl.v)
pal2 <- brewer.pal(8,"Dark2")
png("wordcloud.png", width=800,height=800)
#larger canvas doesn't increase plot size
wordcloud(carl.d$word,carl.d$freq, scale=c(8,.4),min.freq=1,
max.words=Inf, random.order=FALSE, rot.per=.15, colors=pal2)
dev.off()
require(socialR)
upload("wordcloud.png", script="wordcloud.R")
Here’s another way that Google views me: results from the Google citation gadget
Citations for “Carl Boettiger”: 77
Cited Publications: 18
H-Index: 5
Hmm… I think the wordcloud approach might be more accurate… Perhaps no one needed reminding not to trust these kinds of tools, despite the predilection to believe anything quantitative (See the real story on my CV tab (pdf). The other lesson might be that alternative forms of publishing are likely to get picked up by some of these algorithms. Many of the items that come up are talks I’ve deposited on Nature Precedings, and a few are even just notebook posts. Interesting that there’s a growing number of services designed to help scientists strengthen their review dossier using these tools.
Does this mean that alternative publishing (i.e. conference slides, prepints, notebook posts) is a good way to trick the metrics? That people won’t trust the numbers of bloggers, etc, assuming they are inflated? Or that these kinds of communication methods may gain acceptance as part of the scientific discourse?
Citation tools & Future of Publishing [Lab Notebook - R] 2011-02-16 18:04
There has been a lot of rapid development in scientific tools based on a Wordpress platform recently, perhaps spurred in part by the recent Beyond-the-PDF and sessions in Science Online conferences. A new discussion group has emerged around these tools, as described by Martin Fenner.
I have been exploring better tools for citation management within my lab notebook. Recently I have been using the papercite pluginto add citations from my BibTeX files, which are generated from Mendeley, as I described earlier. This has a few performance issues on server load and memory, and a somewhat limited format/appearance options, but more importantly lacks some of the features of being developed in these other citation packages.
To begin, see Martin’s very compelling piece on citations are links. Certainly a link (i.e., to the pdf article) can be part of a citation, but there’s a lot that can be done once we start thinking of the citation itself as a link. Wordpress has a nice interface to handle and organize links, which until now has mostly puzzled me as a overdeveloped blogroll. The Import BibTeX plugin can read my bibliography files and transform each citation into a link in the Wordpress interface, attaching categories corresponding to the source type and to tags I add, (such as to which Mendeley collection the article belongs). Then the Link to Link plugin makes it easy to search and insert these references into my posts. Currently this can’t be done using the bibtex keys, which is a minor bother.
Here’s where things start to get more interesting. The links can embed machine-readable semantic information that adds meta-data to the citation, such as if the citation provides background, methods, supports or disagrees with a claim being made. This can be made visible to the user, but more interestingly this can also be tracked by a computer, which allows for the generation of much more detailed citation statistics. A standard Citation Typing Ontology has been developed and described by David Shotton[cite source=‘doi’]10.1186/2041-1480-1-S1-S6[/cite] and CiTO is now implementedin Link to Link code. While Link to Link seems to have the 10 most common citation types, the ontology provides many more, as listed in the CiTO types documentation.
Finally, the automatic bibliographic information is displayed by the Kcite plugin, currently using the DOI or PMID, which integrates with the Link to Link code. Having installed both plugins and checked the configuration boxes in link to link which allow it to use the kcite shortcode and CiTO syntax, I’m able to highlight some text, select the citation through the search box opened by clicking the Link to Link icon and have it automatically insert the Kcite shortcode using the DOI:
[cite source='doi' rel=cito:cites]10.1186/2041-1480-1-S1-S6[/cite]
At the moment this doesn’t quite work as it seems the rel=cito text disrupts the kcite intepretation of the shortcode. Without this it renders correctly.
Note that in general Link to Link with CiTO works with anything with a URL, and will still embed the CiTO information in the link if no DOI is available, just not add the kcite bibliography.
Also not sure why the kcite bibliography includes the link to the json format, seems it shouldn’t display this. Still active discussion on the mailing list on these topics, and it’s been quite impressive to see how fast development has been moving on these so far.
How to expose/search CiTO meta-data.
kcite option for citing by (Author,year) footnote text, not just by number, like so [1].
Basic fixes: Kcite currently errors with the rel=cito:cites text. Should at least ignore that text until support is built in for CiTO. Apparently link2link should be rendering this with quotes: rel=cito:‘cites’.
toggle json display.
Link2Link requires uploading Bibtex file to add the reference to wordpress link library. Mendeley automatically generates the bibliography – would be great if this could be automatically placed online (i.e. by dropbox) and read in by Wordpress rather than having to manually update. Having to import bibtex every time I add a new article, and then search for the added article via link2link, is rather inefficient. Can merely grab the doi from Mendeley and insert with kcite.
The most obvious advantage of having the references in the Wordpress link library is the ability for link2link to search them and grab the DOI, though searching Mendeley for the DOI is just as easy. So why else would this be useful? A few thoughts:
The link library allows for adding semantic markup to the link, though currently Wordpress does this only for XFN (XHTML Friends Network), designed to describe people. Having CiTO directly in the link editor beside XFN would be nice, though not particularly more functional.
Potential to use blogroll functions of wordpress to display the library listed in the links, though in general Mendeley groups seem a much more powerful way to do this.
Data Mangement on UC3 Merritt Repository [Lab Notebook - R] 2011-02-16 14:06
I’ve been trying to learn a little more about the potential for data management solutions through the UC3 Merritt repository, and decide how this compares to commercial alternatives such as Amazon S3, Picasa/Flickr (obviously for images only), and field-specific and publication specific repositories such as Dryad. Merritt email support have been very helpful in answering my questions and concerns.
The Good: An institutional repository seems like a safer bet than an Internet business, even if it’s as large as Amazon or Google. After all, the UC’s have been around a lot longer.
The Not-So-Good: The big concern for me here is that this is guaranteed (both for IDs and for data itself) only as long as you are (or someone else is) paying the annual fee.
Other Concerns: Can I automate script to upload, download, and embed data? The wealth of tools available for Flickr or Github through the user-extensible API makes it very easy to integrate these repositories into my workflow. I am still unclear how easy this would be with the UC3 archiving services.
Still promising: The staff recognize these challenges, particularly in their contrast to the standard short-term grant-based funding model of science and in working with the research institutions for support covering these costs. Further, it seems the details aren’t set in stone yet, but are an evolving part of the system:
It’s a tough question for which we don’t have a comprehensive answer at the moment. Our fee model does not match up well with the funding model of grant projects–our costs are ongoing, our accounting rules prohibit us from carrying funds across fiscal years, yet our customers may have funding for only a limited time period. We want to partner with organizations committed to the long-term preservation of their digital content. We would work with our partners to find new organizational homes if their commitment changed, due to changes in funding and priorities. This isn’t really an answer, other than to say that we view the relationship as a partnership, and we want to help our partners succeed in their goals for long-term preservation.
–Merritt helpdesk.
The Future of Data: Plans for a UC Davis workshop [Lab Notebook - R] 2011-02-01 04:40
I have been working with several of the faculty of UC Davis to prepare a workshop for our department to (1) help make our researchers aware of the new requirements for NSF proposals and journal submissions with regard to data archiving, (2) To explore what new opportunities this creates and what tools exist that will help our researchers take maximum advantage of it, and (3) to engage our research community in the discussion of future standards for sharing, standardizing, rewarding and funding the archiving of data.
I’m very pleased that we have an excellent panel of supportive faculty:
We are currently working out the format of the workshop. The current draft consists of three 1.5hr sessions, progressing through the three main goals above in an interactive format.
See the recent discussion of these issues in Science, Nature, TREE, Am Nat, and elsewhere. I have started assembling a list of resources in my December notes: Thoughts on the New Policies for Data Archiving at NSF and in Common Journals. In addition, there’s more detail available on the Dryad Wiki and from Dryad here:
Some great information also available from UC3:
In the next month or so we are going to roll out a rule based tool that will ask the researcher a series of questions based on the funding organization that are applying to and will generate a Data Management Plan.
Merritt Repository services, launched in late summer 2010.
EZID Identifier services: obtain and manage long-term identifiers (DOI, ARK) for their digital content. Can assign identifiers to anything: scientific datasets, technical reports, audio files, digital photographs, and non-digital objects as well. This spring the objects with DOIs will appear in Web of Science. (Annual charge unlimited number of DOIs: $75/user, $375/group, or $1,125 for whole university)
Consultation services: Individual consultation, also more workshops on data citation, data sharing, and data rights this Fall.
We have been actively thinking about data and building services that support the management, preservation, and publication of data. We are just about to embark on a project that will create an Excel add-in that will make it easy to archive, share, and publish data embedded in Excel. We are also actively involved in the DataONE project. We recently presented a paper at the Beyond the PDF workshop at UCSD that outlines some of our ideas surrounding data publication.
What would you like to see? Structure, organization, topics?
[iframe http://friendfeed.com/science-2-0/e68945dc/i-m-planning-workshop-at-ucdavis-on-future-of?embed=1 480 400]
Reflections from #scio11: Saturday's Open Science Track [Lab Notebook - R] 2011-01-15 18:12
Have just returned from the amazing and energetic science online 2011 conference, where I will now, like the rest of the attendees, turn to catching up on sleep, following new online acquaintances, struggling not to append #scio11 to everything I write, and composing that nearly obligatory post-event blog entry we all use like a glass of water after a party; with the hope in may help me metabolize the experience.
Never mind that I don’t actually blog. I just have this lab notebook, normally filled with equations, graphs and code. But it is recordings of my research experience, even if it is written only for me most of the time, so let’s just call this part of research and forgive the conversational tone. Meanwhile I will use the excuse to write more at length for myself the notes I wish to remember than would be seemly in writing in a blog with an audience. In the spirit of this resembling my daily notebook and also in actually finishing this post, here are my notes and thoughts on Saturday’s amazing blur. 1
I began the day in Kay’s (@kaythaney) Digital Toolbox session, room C, where I would return for each of my sessions that day. It was one of the live-streamed rooms, so all the following sessions should have archived recordings. The sessions that followed sequentially in this room fit together like a perfectly flowing conversation, as if each one responding to the former and anticipating the next. Just further evidence that Bora ([@Boraz](http://twitter.com/#!/boraz)) and Anton ([@mistersugar](http://twitter.com/#!/mistersugar)) must come from magic lamps.
Next up brought one of my existing luminaries, Steve Koch),2, with Kiyomi Deards and Molly Keener to the panel on data discover-ability and archiving in Open Notebook Science. The session erupted in lively debate on the relative merits of commercial hosting solutions which may offer great discoverability and ease of use until they suddenly go the way of Del.icio.us, or institutional repositories, which are long lived but often unintegrated even amongst themselves. The good-spirited solutions all pointed to doing both and trying to make each better.
Not that we wouldn’t also question that optimism too, for the next session asked us: “What’s keeping us from open science?” Covering many things, at the heart of this session was the crux of lacking incentives. The promise of altmetrics loomed large, but I wondered if we needed to do focus more on making impact with open science than merely measuring it. For instance, we can surely try to measure the impact of open notebooks and science tweets, but tools for better discovery, standardization, or more semantic open content may go much father to having unmistakable impact.
The schedule indicates 3 that lunch happened next. though he whirling discussions left me with lots of ideas and names than memories of food or thoughts to my own upcoming panel. But no matter, I would be sitting with the heroes Jean-Claude Bradley (@jcbradley) and Anthony Williams (@ChemConnector), so why worry?
Our session on Open Notebook Science quickly gave us reason to worry when the projector which had done fine thus far suddenly refused to work. Nevermind, we all work with technology too frequently to be unfamiliar with its failure - we would do without. If you were there and you’re wondering what you missed, here are my slides. See Jean-Claude’s post for more on our session.
I described the tools I use to make my notebook more automated and complete and discussed my vision of a social lab notebook. In face of the observation that many open database efforts face the tragedy of commons effect (many researchers may use the resources but few contribute) I discussed some of the many immediate benefits the practice has had for me. I must say what an immense treat it is to be able to finish a session and see the immediate feedback and reactions it has generated through the online streams.
The video recording of our session has now been posted:
Open Notebook Science: Pushing Data from Bench to Web Service from Smartley-Dunn on Vimeo.
See the recordings of the other sessions as well.
Having broached the idea of altmetrics on several occasions already, our next session dove into the details of how we might construct these, what they might measure, and how they would be received. Or as it was more concisely and provocatively phrased by Paul Groth ([@pgroth](http://twitter.com/#!/pgroth)):
Would you list the number of twitter followers you have on your CV?
The last session dived further into tackling and improving the most common metric: citations. From citations that map to specific parts of articles to ones measuring the sentiment (supports/refutes) or even anticipating the social-cultural context of citations, the potential here is amazing. Jason Hoyt’s ([@jasonHoyt](http://twitter.com/#!/jasonhoyt)) 140 characters may have captured this session best:
if I do my job right, no one will care about publishing in Nature, Science or Cell…
Indicating that better article discovery and citation tracking through tools such as Mendeley would make the impact factor obsolete.
The evening banquet featured many highlights, including seeing one of my fellow graduate students in the video competition, the very moving story of Meg Lowman’s ([@canopymeg](http://twitter.com/#!/canopymeg)) work researching and inspiring rain-forest conservation, laughing tears at Brian Marlow’s ([@sciencecomedian](http://twitter.com/#!/sciencecomedian)) humorist routine, and talks deep into the night about innovated ideas in open education with JC and then the future of publishing with Jason, Mark and Tony ([@jasonPriem](http://twitter.com/#!/jasonpriem), [@science3point0](http://twitter.com/#!/science3point0), and [@ChemConnector](http://twitter.com/#!/ChemConnector)).
Many other great discussions throughout the day on and off line that I cannot begin to capture here. But after all, they are just beginning.
Note this was written up on Jan 17 upon returning to reality with enough time to write, but published under the event date for notebook consistency. Touched up for the Jekyll notebook 2013-07-09, see history in sidebar.↩
pronounced cook, I learned, and all of a sudden understood the chef’s hat symbol for their lab that has puzzled me for a year!↩
Also, the schedule lists the full topics and presenters for these sessions, for which reason I should work in a link to it somewhere.↩
Thoughts on the New Policies for Data Archiving at NSF and in Common Journals [Lab Notebook - R] 2010-12-03 14:08
This post is a work in progress, a scratch pad for me to start assembling what I’ve been learning about and resources pertaining to the new policies emerging from NSF and journals relevant to ecology and evolution. Hoping to highlight not only the policies, but the issues, opportunities, and concerns around them.
I am hoping to help organize a workshop to discuss these issues in my department this Winter. The Davis Open Science group, is planning a series of these workshops, hoping to work with departments, the libraries and the UC Davis Office of Research and its Responsible Conduct of Research program (in compliance with NIH/NSF ethics requirements), as well as resident faculty and editors. Suggestions on what you would like to see in such a workshop much appreciated.
Each discipline has (or will have, see notes and refs by Heather Piwowar it’s own guidelines, but the basic gist is perhaps best summarized in excerpt from this NSF statement:
This supplement should describe how the proposal will conform to NSF policy on the dissemination and sharing of research results (see AAG Chapter VI.D.4 ), and may include:
The types of data, samples, physical collections, software, curriculum materials, and other materials to be produced in the course of the project;
the standards to be used for data and metadata format and content (where existing standards are absent or deemed inadequate, this should be documented along with any proposed solutions or remedies);
policies for access and sharing including provisions for appropriate protection of privacy, confidentiality, security, intellectual property, or other rights or requirements;
policies and provisions for re-use, re-distribution, and the production of derivatives; and
plans for archiving data, samples, and other research products, and for preservation of access to them.
Data management requirements and plans specific to the Directorate, Office, Division, Program, or other NSF unit, relevant to a proposal are available at: http://www.nsf.gov/bfa/dias/policy/dmp.jsp. If guidance specific to the program is not available, then the requirements established in this section apply. [….] The Data Management Plan will be reviewed as an integral part of the proposal, coming under Intellectual Merit or Broader Impacts or both, as appropriate for the scientific community of relevance.
The American Naturalist
This journal requires, as a condition for publication, that data supporting the results in the paper should be archived in an appropriate public archive, such as GenBank, TreeBASE, Dryad, or the Knowledge Network for Biocomplexity. Data are important products of the scientific enterprise, and they should be preserved and usable for decades in the future. Authors may elect to have the data publicly available at time of publication, or, if the technology of the archive allows, may opt to embargo access to the data for a period up to a year after publication. Exceptions may be granted at the discretion of the editor, especially for sensitive information such as human subject data or the location of endangered species.
Whitlock, M.C. et al. Data archiving. The American Naturalist 175, 145-6(2010).
Evolution has a similar policy, outlined in:
Fairbairn, D.J. the Advent of Mandatory Data Archiving. Evolution (2010) (preprint)
Journals with policy in place (links and editions coming)
The American Naturalist (see Whitlock, 2010; above)
Evolution (see Fairbarin, 2010; above)
Journal of Evolutionary Biology (as mentioned in Whitlock)
Molecular Ecology (as mentioned in Whitlock)
Heredity (as mentioned in Whitlock)
ESA journals: Ecology, Ecological Monographs, Ecological Applications, and ** Frontiers**. Currently have a position in Dryad board and a soft data deposition requirement. Also maintains the Ecological Archive. (pers. comm.)
There’s a variety of potential archives. Identifying the best archive for the type of data involved may be complicated.
Cost is a key concern: good archiving takes resources, and does little good if repositories aren’t maintained or persistent. While the repositories are supported by their own grants (for the moment), it seems the repositories will generally charge the journals where the publication accompanying the data will be submitted. The journals may in turn charge the authors, who will have to write these costs into their NSF grants?
Like the policies themselves, these details are still being actively worked out [ref] Todd Vision, associate director of informatics at NESCENT which oversees the Dryad project currently, discusses this in this FF forum. [/ref].
Good references on funding models include these papers by Beagerie and others:
Dryad has a good explanation of what happens when data is deposited.
The need for data archiving in ecology and evolutionary biology has been persuasively argued in articles in a variety of our journals for some time. See:
Moore, A.J. et al. The need for archiving data in evolutionary biology.Journal of evolutionary biology 23, 659-60(2010).
Piwowar, H. a, Day, R.S. & Fridsma, D.B. Sharing detailed research data is associated with increased citation rate. PloS one 2, e308(2007).
In addition to funding, primary concerns involve how data will be shared and cited. Done well, researchers are appropriately accredited and rewarded when there data is re-used, making research faster, supported by larger data sets, more reproducible, and carrying a broader impact. A set of articles highlights many of these issues:
Thorisson, G.A. Accreditation and attribution in data sharing. Nature Biotechnology 27, 984-985(2009).
Sieber, J.E. & Trumbo, B.E. (Not) giving credit where credit is due: Citation of data sets. Science and Engineering Ethics 1, 11-20(1995).
Stodden, V. The Legal Framework for Reproducible Scientific Research: Licensing and Copyright. Computing in Science & Engineering 11, 35-40(2009).
Gleditsch, N.P. & Strand, H. Posting your data: will you be scooped or will you be famous?International Studies Perspectives 4, 89-97(2003).
Vision, T.J. Open Data and the Social Contract of Scientific Publishing. BioScience 60, 330-331(2010).
Costello, M.J. Motivating Online Publication of Data. BioScience 59, 418-427(2009).
Data citation is closely tied to the discussion of data licenses, such as CC licenses that allow conditional reuse, vs content in the public domain that can be used unconditionally. It seems data is not generally viewed by the law as a creative work but as fact, and thus not under the jurisdiction of intellectual property and copyright licenses. Acknowledging this, repositories such as Dryad identify data as Creative Commons zero, which is essentially an intentional decision to put data into the public domain. See more discussion of when this applies and when data can be cc-by, etc, and Wilbanks’ explanation.
The Data Citation collection on Mendeley provides an actively maintained collection of articles discussing practices for depositing, citing, incentives, and tools for data management and publication.
Discussion continues on a few FF forum threads have helped assemble this information:
EDIT
Dryad maintains an excellent list of the growing number of Ecology and Evolution partner journals that mandate archiving
How informative are enrichment analyses really? [Next Genetics » Next Genetics] 2013-09-20 15:49
Enrichment analysis are applied when you have categorical data associated with your dataset. For example gene ontology, pfam families, molecular pathways, enzymatic activity...etc. The gist of the analysis is to see whether a certain category (GO term, pfam…) are over-represented in a subset of your data.
Let’s take an example. Let’s say I have:
What is the significance of this? In other words, if we pick 1,000 genes randomly from the total pool of 20,000 genes, what are the chances there will be more than 300 genes with the cell cycle category?
In this post I will go through the basics of how enrichment analysis is performed and some thoughts on how informative this analysis is as applied to biological systems.
Read MoreClive Brown's talk on the Oxford Nanopore at UKGS2013 [Next Genetics » Next Genetics] 2013-09-02 19:13
I've been attending the UK NGS/Genomic Sciences meetings since it started 4 years ago. While there are great talks every year, this year, they were able to get Clive Brown to do the keynote talk about Oxford Nanopore. For people in the NGS field, I don't think I need to say much about what Nanopore is (check out Oxford Nanopore's website for more details).
Before the talk, Clive put up a slide telling people he prefers there to be no tweets about the talk since he will be covering a great deal of technical details (which he did). I found that kind of strange. It seems like he doesn't want the content of his talk to be public? Why not just have all of us sign a NDA if that's the case? However, I will comply with his request and will not write much about the technical aspects of his talk. Instead, I will talk about what I think about Oxford Nanopore and its potential impact on the field.
Read MorePreview of a very preliminary version of Seeker: Genome Browser [Next Genetics » Next Genetics] 2013-07-07 00:11
I put some finishing touches on Seeker: Annotation Viewer last week for visualizing sequence features such as protein domains, primers, etc... Now I am working on a genome browser. Here is an extremely early prototype (there is around 1.8mb of files to load):
http://www.nextgenetics.net/tools/browser/browser.html
It should work on latest versions of Chrome/Safari/Firefox. It will most likely NOT work on IE or Opera. Hopefully, this won't crash your browser. This is completely client-side only. You can distribute these files on a USB stick and anyone with a modern browser will be able to open it.
The loaded data is human chromosome 1 parsed from a .gtf file downloaded from UCSC. The parsed data is around 1MB (980KB). These interactions are possible right now:
I'll go in to more detail about how the rendering works in the future. I've implemented a "rubber-banding" scrolling system instead of the normal Google Maps style tiling system.
Using webGL to visualize data in the browser [Next Genetics » Next Genetics] 2013-07-01 11:24
I've wanted to learn how to build web apps with webGL ever since I saw the crazy Unreal engine ported to HTML5 and webGL (as a side-note, three.js is a very popular javascript 3d library that leverages webGL). It has a lot of potential for data visualizations. Imagine a genome browser running on a GPU. It will be able to render millions of objects easily.
I came across this developer preview library today of a framework that allows for data visualizations using webGL and webworkers for multi-threading:
http://superconductor.github.io/superconductor/
It is only a developer preview. But it looks extremely cool.
Of course the down-side (as with anything running in a browser) is cross-browser compatbility. The framework seems to also use webCL which doesn't seem like it will be widely adopted anytime soon. Perhaps someone can make a modified Node-webkit?
Annotation viewer finished [Next Genetics » Next Genetics] 2013-06-20 22:02
After several refactoring, version 1.0 of the annotation viewer is finished. You can use the app here:
http://www.nextgenetics.net/tools/anno_view/annotator.html
Input to the app right now is either HMMScan domain table result or a tab delimited file. The tab delimited file is formatted with 5 columns: sequence name, feature name, start position, end position, sequence length. There are sample input data in the app for clarity.
I am not sure how cross-browser it is. It was developed mostly with Chrome in mind, however it should work on latest versions of Chrome/Safari/Firefox.
On the technical side of things, this web app uses D3.js heavily for the SVG rendering and many DOM manipulations. All I can say is that D3.js is almost magical in how fast it re-renders objects. I also rolled my own MVC system instead of going with the popular backbone.js, angular.js,...etc frameworks. It was definintely an eye opening experience to see how much work goes into these MVC systems.
My MVC system is not really a full MVC. A more proper description is a view-centric MVC.
Components like menus, checkboxes, sliders, drop-downs were built with a data binding system that allows them to react to changes in the data. These components are the view of the MVC pattern. However, there are no formal models in this system, hence the "view-centric". Data are just native javascript objects or arrays, allowing JSON-typed input. When the data is bound to a view, methods are added to the data that allows them to update the view on data change. Yes, I am aware that adding methods to the data object is dirty and a hack.
Here is an example of this view-centric MVC system:
var data = {'name':'next gen sequencing conference','attending':false};
var checkbox = new seeker.checkbox()
.bind({'text':data,'checkbox':data},{'text':'name','checkbox':'attending'})
The data is an object consisting of two key:value pairs. To bind this piece of data to a checkbox where the label of the textbox correspond to "name" and the checkbox itself correspond to "attending", we use the .bind function. This function takes in two argument objects: data and keys.
There are specific keys that let's the checkbox component understand which data corresponds to the label or the checkbox. Both the 'text' key which corresponds to the label and the 'checkbox' key which correpsonds to the checkbox are bound to the "data" object. The keys in the object that corresponds to 'text' and 'checbox' are 'name' and 'attending.
This system is a bit unweildy in argument construction. I might have to mess around with that part to make it more elegant. I also still have to optimize data unbinding. I am sure there are tons of memory leaks right now.
Javascript: Offscreen vs CSS display [Next Genetics » Next Genetics] 2013-06-14 12:00
Any developer that deals with a lot of javascript and CSS understands the pain involved in positioning an element exactly right on a page and also have it respond well to window resizes.
A common frustration is creating a "drop-up" menu where a selection menu is displayed above a button on user click. The height of the menu needs to be known to correctly position it above the button. If the menu is dynamically generated, then the height will possibly change everytime the user clicks the button.
The height of the menu can be obtained by getting the element's offset properties (offsetHeight, offsetWidth...). However these properties are only available if the element can be read by the browser screen reader.
A common technique to hide an element while still making it available to the screen reader is to simply move it off the screen:
element.style.left = -10000;
Versus the CSS methodwhich will not make it available to the screen reader.
element.style.display = 'none';
A good overview of various DOM hiding methods can be found here.
I don't really like the offscreen method as it is kind of a hack and I can envision a future scenario where new browser versions will ruin web apps that uses this method. However, this method seems to be the only effective way around this problem.
Is there any performance difference between offscreen and CSS methods? I setup a jsperf test to find out:
http://jsperf.com/offscreen-vs-display-none
I created a new wrapped DOM class and prototyped hide() and offscreen() functions for the CSS and offscreen methods respectively. Then I created 100 wrapped DOM elements as the test setup.
For the two test cases, I either used hide() or offscreen() on the 100 DOM elements. The offscreen() method performs slower than the hide() method by almost 74%. I expected it to be slower as offscreen elements are still being read by the screen reader, but 74% is a pretty large performance hit considering most modern web apps can easily have a few hundred elements.
Javascript: Wrapped vs Extended/Native DOM [Next Genetics » Next Genetics] 2013-06-07 11:25
I've been slowly working on my javascript library for visualizing bioinformatics data. I'v decided not to use too many external frameworks or libraries for two reasons: 1) It is a great learning opportunity for figuring out the nuts and bolts of how javascript runs on browsers. 2) There really aren't that many javascript frameworks designed to handle a lot of data simply because browsers were traditionally not seen as a platform for utilizing tons of data.
There are generally two camps among javascript framework developers for DOM creation and manipulation. One camp extends the DOM by using .prototype function, essentially defining new methods on native element/node objects. The other camp wraps the DOM by creating a new object which in itself, creates a native DOM element/node. All methods of the new object are then applied to the internal DOM element/node.
I prefer wrapped DOM because it offers more flexibility in having private variables and let's me feel secure in not have collisions with native methods. However, the downside to wrapped DOM is the computational overhead that comes with creating every new DOM element/node. How much overhead is it really?
I've setup a quick javascript benchmark where I compare native DOM creation vs wrapped DOM creation with varying prototype methods:
http://jsperf.com/native-dom-versus-wrapped-dom
This test contains 6 cases where the first is native DOM creation with a simple document.createElement(). The other cases are wrapped DOM objects with 1, 5, 10, 15, and 100 prototyped methods. I expected the wrapped DOM to be more expensive than the native object. But do the addition of varying number of prototyped methods matter in object creation?
I get non-consistent results on multiple runs of the test. At worst cases, I am getting around 30% of the performance when creating wrapped DOMs, on best cases, I am getting similar perforamce between native and wrapped DOMs. I am not exactly sure why. It might have something to do with the browser garbage collector as the node might be marked for collection after every iteration. read the update.
However, the differing numbers of prototyped methods doesn't seem to make much of a difference. In fact the object with 100 prototyped methods seemed to perform the best out of the wrapped DOMs in many cases.
Even though a possible 75% 25% decrease in performance is quite a lot, that is still equivalent to 300,000 creations per second, which is probably more than enough for rendering application UI elements. The next step would be to see how wrapped SVG elements perform with native SVG elements....
I've updated the jsperf test to append the new node to the DOM after every creation to make sure it doesn't get collected by the garbage collector. After each case, the new nodes are removed for the next case. Now I seem to be getting more consistent results where wrapped DOM is around 75% of the performance of the native DOM at around 300,000 creations and additions a second.
Working on some web tools for visualizations [Next Genetics » Next Genetics] 2013-05-28 14:02
I am in the middle of my last year as a PhD student, working on my thesis and trying to get some last minute analysis finished. I've also decided to start building up a github profile by working on a few visualization web tools. Hopefully it will add to my CV. The goal of these tools will be to:
The only javascript dependency I'll be using is D3.js.
The first tool I'll be making is an annotation viewer for visualizing features on a set of sequences. Something that's useful for people who want to load 50-100 sequences with protein domain annotations and look at the domain compositions; or for loading various genomic loci with transcript annotations.
I've been working on it past couple of days and I've got most of the feature rendering code finished. You can check out a very preliminary demo here. The sample data is a bunch of SET domain containing genes and PFAM domain annotations:
http://htmlpreview.github.io/?https://github.com/damiankao/seeker/blob/master/test.html
It might not look like much, but coding contextual menus that will respond smartly to edge of the window and refactoring the code for loose coupling took some effort.
It will probably only work on Chrome/Safari/Firefox. There are contextual menus for each feature and sequence for the user to manipulate what features they want shown (left click on features and sequence names). The end goal is to let the user save the rendering as a svg file for further editing.
Here is my github page.
Finding R in a Hadoop World [R-bloggers] 2013-10-31 07:46
by Joseph Rickert
The following is a brief report of all things R encountered in my not quite random, but nevertheless far from determined, walk through the O'Reilly Strata / Hadoop World Conference held this week in NYC. To start off, I had the pleasure of doing a 9:00 AM Monday morning joint tutorial with Antonio Piccolboni, the principal developer of RHadoop, on “Using R and Hadoop for Statistical Computation at Scale”. Antonio began with a no-nonsense presentation of detailed examples showing how to use the functions of the rmr2 and plyrmr packages to write map-reduce jobs. Antonio worked hard for a full two hours going line-by-line through code that performs data reduction and manipulation, cross tabulations and other practical tasks. The audience of 120 plus people stayed right there with Antonio the whole time. The slides including all of the code from Antonio’s tutorial are available on the Conference website.
For my part, I explained the how the parallel external memory algorithms in the newly announced V7 version of Revolution R Enterprise run directly on Hadoop. (My slides are available here.) I have previously posted code giving a preliminary look at how you can go from running algorithms such a as rxLogit, a high performance implementation of logistic regression first on PC and then on a remote Hadoop cluster just by executing a few lines of R code that point to the cluster as a new “compute context”. Nevertheless, seeing things run live makes an impact.
So, why two approaches to running R in Hadoop? Well, why not? A great strength of R is that developers often simultaneously explore alternate approaches to adding some new capability. In this case, I can imagine that data scientists, comfortable with writing map-reduce jobs, might appreciate the option to use plyrmr’s functions to write custom data transformations. On the other hand, statisticians now have the ability to build GLMs, decision trees and other models directly on large samples stored in the HDFS file system of a Hadoop cluster without having to know much about Hadoop at all.
With the dominant focus of the conference being on Hadoop, I didn’t really expect to hear much talk about R. This turned out not to be the case. R has some serious mindshare among the hardcore Hadoop crowd, both in the hallway conversations, and in the presentations too. For example, in this talk: “Hadoop & Data Science for the Enterprise, 30 Tips and Tricks”, Mark Slusar distills a career’s worth of nitty-gritty Hadoop experience into 30 pointed recommendations. In tip number 12 he says straight out: “Use and Learn R packages (they are) huge time-servers”. There is no equivocating here. R is just something you need to know.
Then in a Plenary session talk intended to be provocative, "Beyond R and Ph.D.s : The Mythology of Data Science Debunked”, Douglas Merrill used R as a stand in for the the quantitative aspects of Data Science itself. if nothing else, this serves to establish R’s ubiquity and name recognition in the data science and Hadoop community.
Finally, I had the opportunity to see an R driven demo of the open source H2O software from 0xdata (prounced hexadata) that is making a bit of a splash. 0xdata is a Mountain View-based startup that has implemented a number of impressive statistical and machine learning algorithms (including GBM) in Java to run on Hadoop. The Java algorithms may be accessed by running functions from the h2o R package, wrapper that makes JSON calls to an instance of the H2O software that must be running concurrently with R. The R package and H2O instance may be downloaded from the 0xdata site.
Detecting an Unfair Die with Bayes’ Theorem [R-bloggers] 2013-10-31 00:17
I saw an interesting problem that requires Bayes’ Theorem and some simple R programming while reading a bioinformatics textbook. I will discuss the math behind solving this problem in detail, and I will illustrate some very useful plotting functions to generate a plot from R that visualizes the solution effectively.
The following question is a slightly modified version of Exercise #1.2 on Page 8 in “Biological Sequence Analysis” by Durbin, Eddy, Krogh and Mitchison.
An occasionally dishonest casino uses 2 types of dice. Of its dice, 97% are fair but 3% are unfair, and a “five” comes up 35% of the time for these unfair dice. If you pick a die randomly and roll it, how many “fives” in a row would you need to see before it was most likely that you had picked an unfair die?”
To translate this problem into the language of probability,
We know that
The die that you picked is either fair or unfair, regardless of how many times you roll the die. Thus, given that we observe consecutive fives, a sensible way to decide that a die is unfair is if the probability of the die being fair given
is higher than the probability of the die being unfair given
. Mathematically, we seek the value of
such that
To see why this equation formulates our problem accurately, let’s assume that it is true. Combining Equation and the fact that
is a Bernoulli random variable,
and this is the premise of the question.
To calculate , let’s use Bayes’ Theorem. I always “work out” Bayes’ Theorem by
To apply the definition of conditional probability for our problem,
.
Now, re-write the joint probability in the numerator by applying the definition of conditional probability again, but reversing the conditionality.
.
Finally, apply the law of total probability to the denominator. (Because of the difficulties of writing LaTeX in WordPress, the entire denominator is written on the second line. Please excuse the awkward line placement.)
By Equation , set 0.5 to be less than the above equation.
To solve the problem, we need to find the minimum value of such that Inequality
is true. This inequality cannot be solved analytically, so I computed the right-hand side of
for various possible values of
in R. Below is the resulting plot. In addition to plot(), many functions were used to generate it. I have used text() to add custom text inside the plot, abline() to add a custom line, and axis() to add custom tick marks before, but this is the first time that I have used mtext() to add a custom tick label to the vertical axis. I encourage you to study the code carefully to learn all of the useful functions and build your own plot with custom axes, tick marks, and tick labels.
Notice that I needed to use the axis() function twice to add all of the tick marks that I wanted. For some reason, I could not add ’0′ and ’1′ among the first set of tick marks, but I could add them separately in a second calling of axis().
I also found a document by Tian Zheng from Columbia University that lists the names of various colours in R. It turns out that there are many more colours than the standard ones that I usually use from the colours of a rainbow!
As the plot shows, if you see 5 or more consecutive “fives”, you have reason to suspect that the die is unfair.
##### Detecting an Unfair Die with Bayes' Theorem
##### By Eric Cai - The Chemical Statistician
# set the possible values of the number of consecutive "fives" observed from rolling a die
x = 1:20
# calculate the probability of the die being unfair given the observed number of consecutive "fives"
prob.loaded = (0.35^x)*0.03/((0.35^x)*0.03 + ((1/6)^x)*0.97)
# export the plot as a PNG image file
png('INSERT YOUR DIRECTORY PATH HERE/unfair die plot.png')
# plot the calculated probabilities
# notice that the "yaxt = 'n'" option suppresses the verticle axis; I want to add my own custom axis later
plot(x, prob.loaded, col=ifelse(x == 5, "forestgreen", "black"), ylab = 'P(Die is Fair | X)', ylim = c(-0.05, 1.05), xlab = 'X - Number of Consecutive Fives Observed', yaxt='n', main = "Using Bayes' Theorem to Detect an Unfair Die")
# add a custom horizontal line to show where P(Die = Unfair | X = x) = 0.5
abline(0.5, 0, col = 'firebrick1')
# add a custom tick mark with the colour 'firebrick1'
# the label is left intentionally blank, because its colour can only be black
# use mtext() to set the tick label's colour
axis(2, at = c(0.5), col = 'firebrick1', labels = ' ')
# add a custom tick label at P(Die = Unfair | X = x) = 0.5
# distinguish it with the colour 'firebrick1'
mtext(text = '0.5', side = 2, line = 1, col = "firebrick1")
# add text to denote the point of interest
text(9, 0.55, 'P(Die is loaded | X = 5) = 0.5581', col = 'forestgreen')
# add some other tick marks to show the other values along the vertical axis
axis(2, at = c(0.1, 0.3, 0.7, 0.9), labels = c('0.1', '0.3', '0.7', '0.9'))
axis(2, at = c(0, 1), labels = c('0', '1'))
dev.off()
Durbin, R. (Ed.). (1998). Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge university press.
What Hadley Wickham uses [R-bloggers] 2013-10-30 18:41
You know Hadley Wickham as the inventor of the ggplot2 visualization phenomenon, the creator of time-saving R packages like plyr and lubridate, and the Chief Scientist at RStudio. But do you know what laptop Hadley uses, what software he uses (besides, R, of course), or his favourite kitchen appliance? Find out Hadley's interview with The Setup. (Also check out our profile of Hadley from September 2010.)
The Setup: Hadley Wickham
R and my divorce from Word [R-bloggers] 2013-10-30 15:10

Being in grad school, I do a lot of scholarly writing that requires associated or embedded R analyses, figures, and tables, plus bibliographies.
Microsoft Word makes this unnecessarily difficult.
Many tools are now available to break free from the tyranny of Word.
The ones I like involve writing an article in markdown format, integrating all data preparation, analysis, and outputs with the document (e.g. with the excellent and accessible knitr package or with a custom make set up like this one).
Add in version control with Git, and you’ve got a nice stew going.
If you’re involved in the open source/reproducible research blogo-twittersphere, this is probably old hat. To many in my department, this looks like black magic. It’s not.
I can’t give an authoritative overview, but here are some resources that helped me get through my divorce:
Binomial confidence intervals: exact vs. approximate [R-bloggers] 2013-10-30 15:04
Financial Data Accessible from R – part II [R-bloggers] 2013-10-30 04:44
I updated my initial post with two new sources of data and the associated R packages: Datastream and PWT. I also added the fImport package from Rmetrics. Following a reader suggestion, I made the initial table more interactive, moved the data description and package detail below the main table and updated them.
Enjoy!
| Source | R Package | Free Access | Available on CRAN | Package/Data URL |
|---|---|---|---|---|
| Yahoo, FRED, Oanda, Google | Quantmod | Yes | Yes | http://www.quantmod.com/ |
| Quandl | Quandl | Yes | Yes | http://www.quandl.com/help/packages/r |
| TrueFX | TFX | Yes | Yes | http://rpubs.com/gsee/TFX |
| Bloomberg | Rbbg | No | No | http://findata.org/rbloomberg/ |
| Interactive Broker | IBrokers | No | Yes | https://www.interactivebrokers.com/en/main.php |
| Datastream | rdatastream | No | No | https://github.com/fcocquemas/rdatastream |
| Penn World Table | pwt | Yes | Yes | https://pwt.sas.upenn.edu/ |
| Yahoo, FRED, Oanda | fImport | Yes | Yes | http://www.rmetrics.org/ |
Data Description
Package Detail
Percolation Threshold on a Square Lattice [R-bloggers] 2013-10-30 01:38
Manfred Schroeder touches on the topic of percolation a number of times in his encyclopaedic book on fractals (Schroeder, M. (1991). Fractals, Chaos, Power Laws: Minutes from an Infinite Paradise. W H Freeman & Company.). Percolation has numerous practical applications, the most interesting of which (from my perspective) is the flow of hot water through ground coffee! The problem of percolation can be posed as follows: suppose that a liquid is poured onto a solid block of some substance. If the substance is porous then it is possible for the liquid to seep through the pores and make it all the way to the bottom of the block. Whether or not this happens is determined by the connectivity of the pores within the substance. If it is extremely porous then it is very likely that there will be an open path of pores connecting the top to the bottom and the liquid will flow freely. If, on the other hand, the porosity is low then such a path may not exist. Evidently there is a critical porosity threshold which divides these two regimes.
This situation can be modelled on a regular lattice where each of the lattice sites is either occupied (with probability p) or vacant (with probability 1-p). This is known as the site percolation problem. The related bond percolation problem can be posed in terms of whether or not the edges between neighbouring sites are open or closed. As we will see there is a well defined range of p (centred on a threshold value pc) for which the probability of percolation decreases rapidly from one to zero.
The percolation problem has been studied on a menagerie of lattice types but we will focus on the simplest, the square lattice.
First we will load a few handy libraries and set up some constants.
> library(ggplot2) > library(reshape2) > library(plyr) > > EMPTY = 0 > OCCUPIED = 1 > FLOW = 2
Next a function to generate and populate a lattice, which takes as arguments the width of the lattice and the occupation probability.
> create.grid <- function(N, p) {
+ grid = matrix(rbinom(N**2, 1, p), nrow = N)
+ #
+ attributes(grid)$p = p
+ #
+ return(grid)
+ }
>
> set.seed(1)
> g1 = create.grid(12, 0.6)
> g2 = create.grid(12, 0.4)
> g1
[,1] [,2] [,3] [,4] [,5] [,6] [,7] [,8] [,9] [,10] [,11] [,12]
[1,] 1 0 1 0 0 0 1 0 1 0 0 1
[2,] 1 1 1 1 0 1 1 1 1 1 1 0
[3,] 1 0 1 0 1 1 1 0 0 0 1 0
[4,] 0 1 1 1 0 1 0 1 0 0 1 1
[5,] 1 0 0 0 1 0 0 1 0 1 0 1
[6,] 0 0 1 0 1 1 1 1 1 1 1 1
[7,] 0 1 1 0 1 1 0 1 1 1 1 0
[8,] 0 0 1 1 1 0 0 1 0 1 1 1
[9,] 0 0 1 1 1 1 1 0 0 0 1 0
[10,] 1 1 1 0 1 0 0 0 1 1 1 0
[11,] 1 0 0 1 0 1 1 0 1 1 1 1
[12,] 1 1 0 1 1 0 1 0 1 0 1 1
In the text representation of the lattice the occuppied sites are represented by a 1, while a 0 indicates a vacant site.
> visualise.grid <- function(g) {
+ N = nrow(g)
+ #
+ limits = c(0, N) + 0.5
+ #
+ ggplot(melt(g[nrow(g):1,], 1, varnames = c("row", "col"), value.name = "occupied")) +
+ geom_tile(aes(x = col, y = row, fill = factor(occupied))) +
+ geom_hline(yintercept = limits, size = 2) +
+ geom_vline(xintercept = limits, size = 2) +
+ xlim(limits) + ylim(limits) +
+ coord_fixed(ratio = 1) +
+ scale_fill_manual(values = c("white", "grey", "lightblue")) +
+ theme(panel.background = element_blank(),
+ axis.title = element_blank(),
+ axis.text = element_blank(),
+ axis.ticks = element_blank(),
+ legend.position = "none"
+ )
+ }
>
> require(gridExtra)
>
> grid.arrange(visualise.grid(g1), visualise.grid(g2), ncol = 2)
A graphical representation makes things a lot clearer.
Now we need to check for flow through the lattice. We will use a recursive depth first search. Everything goes into a single function. The first argument is the grid. The second and third (optional) arguments are the row and column indices for a particular cell in the grid. If the latter arguments are omitted then the function loops through each of the cells in the top row of the grid. If a particular cell is specified, and it is not already occupied, then it is marked as being part of a flow path. The function then recursively considers each of the nearest neighbour cells.
> flow <- function(g, i = NA, j = NA) {
+ # -> Cycle through cells in top row
+ #
+ if (is.na(i) || is.na(j)) {
+ for (j in 1:ncol(g)) {
+ g = flow(g, 1, j)
+ }
+ return(g)
+ }
+ #
+ # -> Check specific cell
+ #
+ if (i < 1 || i > nrow(g) || j < 1 || j > ncol(g)) return(g)
+ #
+ if (g[i,j] == OCCUPIED || g[i,j] == FLOW) return(g)
+ #
+ g[i,j] = FLOW
+
+ g = flow(g, i+1, j) # down
+ g = flow(g, i-1, j) # up
+ g = flow(g, i, j+1) # right
+ g = flow(g, i, j-1) # left
+
+ g
+ }
The flow path can then be visualised.
> grid.arrange(visualise.grid(flow(g1)), visualise.grid(flow(g2)), ncol = 2)
The grid on the left does not percolate while the one on the right does.
Finally we can determine whether a given grid percolates by checking whether or not the flow makes it to the bottom of the grid.
> # Check whether flow reaches to bottom of the lattice.
> #
> percolates <- function(g) {
+ g <- flow(g)
+ for (j in 1:ncol(g)) {
+ if (g[nrow(g), j] == FLOW) return(TRUE)
+ }
+ return(FALSE)
+ }
>
> percolates(g1)
[1] FALSE
> percolates(g2)
[1] TRUE
Finding the percolation threshold is a little numerically intensive, so it makes sense to take advantage of parallel execution.
> library(foreach) > library(doMC) > > registerDoMC(cores=4)
We define some parameters for the calculation: the dimensions of the lattice and the number of replicates for each value of the occupation probability, p.
> N = 25 > REPLICATES = 1000
Although we will be considering the full range of possible values for p, it makes sense to focus our attention on those values closer to the threshold. For each value of p we randomly generate a number of grids and check whether or not each one percolates.
> pseq = unique(c(seq(0, 0.2, 0.1), seq(0.2, 0.6, 0.0125), seq(0.6, 1, 0.1)))
> #
> perc.occp = foreach(p = pseq, .combine=rbind) %dopar% {
+ data.frame(p, percolates = replicate(REPLICATES, percolates(create.grid(N, p))))
+ }
Summary statistics are then compiled to give the percolation probability as a function of p.
> perc.summary = ddply(perc.occp, .(p), summarize, + mean = mean(percolates), + sd = sd(percolates) / sqrt(length(percolates)) + )
A logistic model is fitted to these data and the threshold value for p is estimated.
> logistic.fit = glm(mean ~ p, family=binomial(logit), data = perc.summary)
> #
> perc.summary$fit = logistic.fit$fitted
> #
> (pcrit = - logistic.fit$coefficients[1] / logistic.fit$coefficients[2])
(Intercept)
0.41026
> 1-pcrit
(Intercept)
0.58974
That compares pretty well with the reference results for the 44 square lattice.
Finally, all of this is put together in a plot.
> ggplot(perc.summary, aes(x = p, y = mean)) +
+ geom_point(size = 3, shape = 21) +
+ geom_line(aes(x = p, y = fit), linetype = 2) +
+ geom_vline(xintercept = pcrit, color = "blue") +
+ annotate("text", label = sprintf("p1 == %.5f", pcrit), x = 0.9, y = 1.0, parse = TRUE) +
+ scale_x_continuous(breaks = seq(0, 1, 0.1)) +
+ scale_y_continuous(breaks = seq(0, 1, 0.2)) +
+ ylab("Percolation Probability") +
+ theme_classic()
swirl: Learning Statistics & R [R-bloggers] 2013-10-29 22:53
Three jobs at Monash [Hyndsight] 2013-10-18 00:02
We are currently advertising for three academic positions, suitable for recent PhD graduates.
Please don’t send any questions to me. Click the “More information” links and follow the instructions.
Probabilistic Energy Forecasting [Hyndsight] 2013-10-14 00:07
The International Journal of Forecasting is calling for papers on probabilistic energy forecasting. Here are the details (taken from Tao Hong’s blog).
In today’s competitive and dynamic environment, more and more decision making processes in the energy industry are relying on probabilistic forecasts. The applications of probabilistic energy forecasts spread across planning and operations of the entire energy value chain. We are seeking papers from researchers working on the areas of probabilistic energy forecasting. “Energy forecasting” refers to “forecasting in the energy industry”, which includes but is not limited to forecasting supply, demand and price of electricity, gas, water, and renewable energy resources. The probabilistic forecasts can take various forms, e.g., from quantile to full density forecasts, probabilistic forecasts for multi-categorical variables or functional data.
The potential topics include:
If you are interested in contributing a paper to this special issue, please submit a one-page extended abstract with a cover letter to both editors for an initial review. Authors of selected abstracts will be invited to submit full papers to the International Journal of Forecasting. In addition, winners of the Global Energy Forecasting Competition 2014 (www.gefcom.org) will be invited to contribute to this special issue as well.
The proposed schedule is listed below:
Prof. Tao Hong, University of North Carolina at Charlotte, USA (hong@uncc.edu)
Prof. Pierre Pinson, Technical University of Denmark, Denmark (ppin@elektro.dtu.dk)
Robert G Brown (1923–2013) [Hyndsight] 2013-10-09 07:52
Robert Goodell Brown was the father of exponential smoothing. He died last week at the age of 90. While I never met him, I was indebted to him for exponential smoothing and his practical and insightful books.
Today I received this email from King Harrison III advising of his death.
Twenty years ago I attended the ISF 93 conference in Pittsburgh, which honored Bob Brown on his 70th birthday, and his contributions to the forecasting world.
My reason to attend ISF 93? To meet the man who had developed Exponential Smoothing, which I had been using in inventory replenishment systems for years.
That began a 20 year relationship with Bob, first in a business context, and then the past 8 years as a friend.
He passed last week, at home.
I attended the memorial service for Bob this past Sunday, at his home in Vermont.
This morning his son Todd Brown sent Bob’s obit, at the link below.
http://www.vnews.com/obituaries/8832880–95/robert-goodell-brown
For the past 2 decades Bob has been my mentor on both business and life challenges. I will miss him.
At my last annual visit to see Bob, this past June, he asked if I was interested in his working papers and research material for his career.
The first 200 lbs of that information arrived a month ago, and is in my office.
His son Todd is working with me to obtain the remaining information.
I have contacted Georgia Tech as a landing place for all of Bob’s work.
If you have questions, certainly contact me at the information below.
Best regards,
King III (king3@k3s.com)
Questions on my online forecasting course [Hyndsight] 2013-10-03 21:56
I’ve been getting emails asking questions about my upcoming course on Forecasting using R. Here are some answers.
Open source R is fine. Revolution Analytics is organizing the course, but there is no requirement to use their software. I will be using open source R with Rstudio for demonstrating things in lectures.
No. It costs $1000 to register for the course. The course is being run jointly by Revolution Analytics and Monash University. Most of the money will go to Monash University, and will fund a research assistant to help me with R package development. My R packages and my FPP textbook are free, but they do take time and resources to produce. This course is one way I am funding them.
Provided you are registered, you will be able to log in and watch the lectures after the event. If you really want to, you can watch them repeatedly. This is particularly important for people in Eastern Europe or West Asia where the lectures are in the middle of the night.
Yes. The lectures are meant to be interactive, for those watching live. You can ask questions, interject, etc. I will also try to respond to questions after lectures, especially for those who cannot watch live.
Probably. The course is based on my FPP book, so don’t expect me to cover things that aren’t discussed there. But most people find that they learn more through asking questions, seeing examples worked out, discussion, etc.
It is worth brushing up on R if you don’t use it regularly. I will not be using Rcmdr. Instead, I will use Rstudio and we will be learning the relevant commands. A suitable intro is the tutorial at www.otexts.org/fpp/using-r.
If you feel that is not enough, try working through the tutorials at cyclismo.org.
The course is not difficult from a mathematical/statistical perspective, and as long as you are not freaked out by a few equations you should be fine. If you wanted to brush up, go over multiple regression at an introductory level. A suitable background on this is chapter 5 of FPP, or chapter 3 of Pardoe’s Applied regression modeling. But you may have something else handy that would be equally suitable.
If you really want to get a head start, try working through Michael Lundholm’s tutorial on R’s time series facilities.
The course is about time series forecasting. That is, when you want to forecast data collected regularly over time. So if you have annual profits, or monthly sales data, or weekly electricity demand, or daily passenger numbers, or something else collected regularly over time, then this course should be helpful. But if your forecasting problem does not fit into that paradigm, then it is probably not the course you need.
OTexts.org is launched [Hyndsight] 2013-09-26 19:08
The publishing platform I set up for my forecasting book has now been extended to cover more books and greater functionality. Check it out at www.otexts.org.
So far, we have three complete books:
and one book currently being written:
Thanks to Souhaib Ben Taieb for doing a lot of the work to make OTexts a reality. I’ve talked about an online textbook publishing platform for several years, and so it is great to finally see it happening.
There is a lot more to come. We hope to add new books, and new features. The new features include:
Please check out the new site, and if you’re a potential author, please contact us. As you will see from what we already have online, LaTeX is no problem (we are using MathJax as the LaTeX renderer). But we haven’t yet started making the most of the dynamic online platform by having multimedia content, interactive content, and so on. Hopefully some new authors will help us to reimagine what a modern textbook can be!
Forecasting with R [Hyndsight] 2013-09-25 19:10
The following video has been produced to advertise my upcoming course on Forecasting with R, run in partnership with Revolution Analytics.
The course will run from 21 October to 4 December, for two hours each week. More details are available at http://robjhyndman.com/hyndsight/revolutionr2013/
If you are interested, please register for the course at the following link.
Forecasting with daily data [Hyndsight] 2013-09-16 20:26
I’ve had several emails recently asking how to forecast daily data in R. Unless the time series is very long, the easiest approach is to simply set the frequency attribute to 7.
y <- ts(x, frequency=7) |
Then any of the usual time series forecasting methods should produce reasonable forecasts. For example
library(forecast) fit <- ets(y) fc <- forecast(fit) plot(fc) |
When the time series is long enough to take in more than a year, then it may be necessary to allow for annual seasonality as well as weekly seasonality. In that case, a multiple seasonal model such as TBATS is required.
y <- msts(x, seasonal.periods=c(7,365.25)) fit <- tbats(y) fc <- forecast(fit) plot(fc) |
This should capture the weekly pattern as well as the longer annual pattern. The period 365.25 is the average length of a year allowing for leap years. In some countries, alternative or additional year lengths may be necessary. For example, with the Turkish electricity data analysed in De Livera et al (JASA 2011), we used three seasonal periods: 7, 354.35 and 365.25. The period 354.37 is the average length of the Islamic calendar.
Capturing seasonality associated with moving events such as Easter or the Chinese New Year is more difficult. Even with monthly data, this can be tricky as the festivals can fall in either March or April (for Easter) or in January or February (for the Chinese New Year). The usual seasonal models don’t allow for this, and even the complex seasonality discussed in my JASA paper assumes that the seasonal patterns occur at the same time in each year. The best way to deal with moving holiday effects is to use dummy variables. However, neither ETS nor TBATS models allow for covariates. A state space model of the same form as TBATS but with multiple sources of error and covariates could be used, but I don’t have any R code to do that.
Instead, I would use a regression model with ARIMA errors, where the regression terms include any dummy holiday effects as well as the longer annual seasonality. Unless there are many decades of data, it is usually reasonable to assume that the annual seasonal shape is unchanged from year to year, and so Fourier terms can be used to model the annual seasonality. Suppose we use
Fourier terms to model annual seasonality, and that the holiday dummy variables are in the vector holiday with 100 future values in holidayf. Then the following code will fit an appropriate model.
y <- ts(x, frequency=7) z <- fourier(ts(x, frequency=365.25), K=5) zf <- fourierf(ts(x, frequency=365.25), K=5, h=100) fit <- auto.arima(y, xreg=cbind(z,holiday)) fc <- forecast(fit, xreg=cbind(zf,holidayf), h=100) |
The order
can be chosen by minimizing the AIC of the fitted model.
MAXIMA research centre at Monash Uni [Hyndsight] 2013-09-10 21:17
The “Monash Academy for Cross and Interdisciplinary Mathematical Applications” (MAXIMA) is a new research centre that aims to maximise the potential of mathematics to deliver impact to society. It will be led by Kate Smith-Miles. I will also be involved along with several other mathematicians at Monash. Our mission at MAXIMA is to find solutions to 21st century problems by dismantling mathematical barriers.
MAXIMA will be launched on 25 September at a public lecture on “The Role of Embedded Optimization in Smart Systems and Products”.
More details at community.monash.edu/maxima
Online course on forecasting using R [Hyndsight] 2013-09-10 20:13
I am teaming up with Revolution Analytics to teach an online course on forecasting with R. Topics to be covered include seasonality and trends, exponential smoothing, ARIMA modelling, dynamic regression and state space models, as well as forecast accuracy methods and forecast evaluation techniques such as cross-validation. I will talk about some of my consulting experiences, and explain the tools in the forecast package for R.
The course will run from 21 October to 4 December, for two hours each week. Participants can network and interact with other practitioners through an online community.
The following video provides an introduction to the course:
And these slides give a few more details:
I will assume you already know how to use R. If you don’t, you can quickly get up to speed by doing a few online tutorials.
I will also assume that you know some basic statistics, including multiple regression. You can brush up by reading this chapter from my online book. However, you will not need to know any time series or forecasting.
If you are interested, please register for the course at the following link.
Some questions about the course are answered here.
Reflections on UseR! 2013 [Hyndsight] 2013-07-12 18:01
This week I’ve been at the R Users conference in Albacete, Spain. These conferences are a little unusual in that they are not really about research, unlike most conferences I attend. They provide a place for people to discuss and exchange ideas on how R can be used.
Here are some thoughts and highlights of the conference, in no particular order.
ets() function in the forecast package. Christoph is also responsible for the big improvement in speed of the ets() function from v4.05 of the forecast package.Thanks to the committee and sponsors for making it a great conference. The next one is in California: UseR! 2014.
Why I am I growing worried about reproducibility? [Digital Notebook - Vince Buffalo] 2012-03-25 03:00
Why I am I growing worried about reproducibility? For three reasons:
Reproducibility is a fringe movement in the sciences now.
Reproducibility is hard.
3.
Right now, I could walk to the hardware store, buy some pea seeds, and replicate genetics research that’s 150 years old. I could also replicate just Mendel’s analysis with his raw data. However, I can’t
Using Bioconductor to Analyze your 23andme Data [Digital Notebook - Vince Buffalo] 2012-03-12 03:00
This is a working draft; I may add to this in the future.
Bioconductor is one of the open source projects of which I am most fond. The documentation is excellent, the community wonderful, the development fast-paced, and the software very well written. I’ll introduce the power of Bioconductor while exploring some raw SNP data from 23andme.com.
I’ll be using a new package in the development branch (due to be released with Bioconductor version 2.10 very soon) called gwascat. gwascat is a package that serves as an interface to the NHGRI’s database of genome-wide association studies. I’ll also be using the development version of AnnotationDbi, which you’ll need to grab if you want to follow along.
If you’re reading this after the release of 2.10, use biocLite to download; otherwise install the source package via R CMD INSTALL. Loading the package with library(gwascat) creates a GRanges instance of SNPs and their diseases. GRanges is a fundamental data structure in Bioconductor (specifically the GenomicRanges package) that is designed to hold ranges on genomes efficiently, as well as metadata about the ranges. In this case, the object gwrngs holds SNP ranges (well, locations) and metadata provided by the GWA studies in NHGRI’s database.
While 23andme provides a great interface to one’s genotyping information and GWA research, Bioconductor offers a different way to explore and learn from your genotyping data.
When I was considering 23andme, I ultimately persuaded by the fact that they release their raw genotype calls to users. Unfortunately they do so without SNP call confidence data, but in a personal correspondence with a 23andme representative they stated:
Data reproducibility of our genotyping platforms is estimated at about 99.9%. Average call rate is about 99%. When samples do not meet sufficient call rate thresholds, we repeat the analysis, and/or request a new sample. We do not return data to customers that does not meet our quality thresholds.
Now, 99.9% sounds like a lot, but considering there are 960,545 SNPs being called, it’s not that high.
To retrieve raw data, simply click the “Account” link at the top of the page (after you’ve signed in) and click “Browse Raw Data”. There should be a download link. If you’ve never used GPG to encrypt a file, now is the time to learn; keep your SNP data encrypted.
The file 23andme provides has four columns: rs ID, chromosome, position, and genotype.
Use read.table to load this data in R. It’s a lot of data, so providing this function with information about the type of data can speed this up quite a bit. Here is the code I used:
d <- read.table("data/genome_Vince_Buffalo_Full_20120313162059.txt", sep="\t",
colClasses=c("character", "character", "numeric", "character"),
col.names=c("rsid", "chrom", "position", "genotype"),
header=FALSE)
You may notice that chromosome has the class “character” - this is because there are chromosomes X, Y, and MT (for mitochondrial). For later plotting purposes, it’s good to make this an ordered factor:
tmp <- d$chrom
d$chrom = ordered(d$chrom, levels=c(seq(1, 22), "X", "Y", "MT"))
## It's never a bad idea to check your work
stopifnot(all(as.character(tmp) == as.character(d$chrom)))
Using Hadley Wickham’s excellent ggplot2 package, we can look at the distribution of SNPs by chromosome:
ggplot(d) + geom_bar(aes(chrom))

This isn’t providing information on SNP density as much as it is chromosome length (except X). We’ll take a more detailed look a bit later.
Another really wonderful aspect of Bioconductor is that the project isn’t just a repository of code: it also stores annotation, full genomes, and experimental data. Such packaged data is the foundating of reproducible bioinformatics, as you no longer have to worry about keeping track of data versions and storing downloaded data yourself. If you need to work with cutting edge data from Ensembl or UCSC tracks, the packages biomaRt and rtracklayer work well.
Suppose I want to see if any of my SNPs fall in the APOE gene region. For this, I’ll need transcript annotation data, as this tells me the range and chromosome of this gene. If I wished to create a fresh database of exon, gene, transcript, and splicing data, I could with the GenomicFeature package. This package has methods for building transcriptDb objects from the Known Gene track from UCSC, as well as Ensembl databases. However, I’ll just use a pre-packaged version, TxDb.Hsapiens.UCSC.hg18.knownGene. I use hg18 rather than hg19 because this is the build that 23andme’s coordinates reference.
library(TxDb.Hsapiens.UCSC.hg18.knownGene)
txdb <- TxDb.Hsapiens.UCSC.hg18.knownGene
class(txdb) ## do some digging around!
transcriptDb objects have nice accessor functions for accessing their components. Behind the scenes, everything is in SQLite and very efficient (are you seeing why I love Bioconductor?).
If we look at the transcripts with the transcripts accessor function, we see it’s a GenomicRanges object:
transcripts(txdb)
GRanges with 66803 ranges and 2 elementMetadata values:
seqnames ranges strand | tx_id tx_name
<Rle> <IRanges> <Rle> | <integer> <character>
[1] chr1 [ 1116, 4121] + | 1 uc001aaa.2
[2] chr1 [ 1116, 4272] + | 2 uc009vip.1
[3] chr1 [ 19418, 20957] + | 26 uc009vjg.1
[4] chr1 [ 55425, 59692] + | 28 uc009vjh.1
[5] chr1 [ 58954, 59871] + | 29 uc001aal.1
[6] chr1 [310947, 310977] + | 33 uc001aaq.1
[7] chr1 [311009, 311086] + | 34 uc001aar.1
[8] chr1 [314323, 314353] + | 35 uc001aas.1
[9] chr1 [314354, 314385] + | 36 uc001aat.1
... ... ... ... ... ... ...
[66795] chrY [25318610, 25368905] - | 33721 uc004fwl.1
[66796] chrY [25318610, 25368905] - | 33722 uc010nxm.1
[66797] chrY [25586438, 25607639] - | 33731 uc004fws.1
[66798] chrY [25739178, 25740308] - | 33732 uc004fwt.1
[66799] chrY [25949151, 25949179] - | 33733 uc004fwu.1
[66800] chrY [26012854, 26012887] - | 33734 uc004fww.1
[66801] chrY [26015033, 26015066] - | 33735 uc004fwx.1
[66802] chrY [26015782, 26015809] - | 33737 uc004fwy.1
[66803] chrY [26016792, 26016820] - | 33738 uc004fwz.1
To interact with the wealth of data behind a transcriptDb object, we often group individual ranges into groups, leaving us with a GRangesList.
tx.by.gene <- transcriptsBy(txdb, "gene")
tx.by.gene
GRangesList of length 20121:
$1
GRanges with 2 ranges and 2 elementMetadata values:
seqnames ranges strand | tx_id tx_name
<Rle> <IRanges> <Rle> | <integer> <character>
[1] chr19 [63549984, 63556677] - | 61027 uc002qsd.2
[2] chr19 [63551644, 63565932] - | 61033 uc002qsf.1
$10
GRanges with 2 ranges and 2 elementMetadata values:
seqnames ranges strand | tx_id tx_name
[1] chr8 [18293035, 18303003] + | 26503 uc003wyw.1
[2] chr8 [18301794, 18302666] + | 26504 uc010lte.1
$100
GRanges with 2 ranges and 2 elementMetadata values:
seqnames ranges strand | tx_id tx_name
[1] chr20 [42681577, 42713790] - | 62142 uc002xmj.1
[2] chr20 [42681577, 42713790] - | 62143 uc010ggt.1
...
<20118 more elements>
Holy GRangeList batman! These are the transcripts grouped by gene. There are other methods for grouping by CDS and exons (cdsBy and exonsBy).
The names of the list elements are Entrez gene IDs. We can look up specific genes with another Bioconductor annotation package, org.Hs.eg.db. There are org.* annotation packages for many organisms. You can forge your own and interact with them with the AnnotationDbi package. I’m using a development version of this package that has a new slick SQL-like interface; it will be widely available with the upcoming 2.10 release.
Suppose I want to convert the Entrez Gene IDs to gene names. The “eg” in org.Hs.eg.db refers to Entrez Gene IDs. Printing the org.Hs.eg.db object gives a nice list of information. Let’s look for the APOE gene’s Entrez Gene ID.
library(org.Hs.eg.db)
cols(org.Hs.eg.db)
[1] "ENTREZID" "ACCNUM" "ALIAS" "CHR" "ENZYME"
[6] "GENENAME" "MAP" "OMIM" "PATH" "PMID"
[11] "REFSEQ" "SYMBOL" "UNIGENE" "CHRLOC" "CHRLOCEND"
[16] "PFAM" "PROSITE" "ENSEMBL" "ENSEMBLPROT" "ENSEMBLTRANS"
[21] "UNIPROT" "UCSCKG" "GO"
These are the columns we can query out. Certain keys exist: we can access these using keytypes(). Using it all together, we can extract the Entrez Gene ID:
select(org.Hs.eg.db, keys="APOE", cols=c("ENTREZID", "SYMBOL", "GENENAME"),
keytype="SYMBOL")
SYMBOL ENTREZID GENENAME
APOE 348 apolipoprotein E
Now, we can look for this in our tx.by.gene GRangesList. A word of caution: Entrez Gene IDs are names and thus they need to be quoted when working with GRangesList objects from transcript databases.
tx.by.gene["348"]
GRangesList of length 1:
$348
GRanges with 1 range and 2 elementMetadata values:
seqnames ranges strand | tx_id tx_name
<Rle> <IRanges> <Rle> | <integer> <character>
[1] chr19 [50100879, 50104490] + | 59642 uc002pab.1
If I had used tx.by.gene[348] the 348th element of the list would have been returned, not the transcript data for the APOE gene (which has Entrez Gene ID “348”).
Now, do any SNPs fall in this region? Let’s build a GRanges object from my genotyping data, and look for overlaps. Before I do, it’s worth mentioning another gotcha about working with bioinformatics data: chromosome naming schemes. Different databases use all sorts of schemes, and you should always check them. 23andme returns just numbers, X, Y, and MT. Let’s change it to use the same as the Bioconductor annotation.
# CAREFUL: use levels() to check that you're making new factor names
# that correspond to the old ones!
levels(d$chrom) <- paste("chr", c(1:22, "X", "Y", "M"), sep="")
my.snps <- with(d, GRanges(seqnames=chrom,
IRanges(start=position, width=1),
rsid=rsid, genotype=genotype)) # this goes into metadata
Now, let’s find overlaps using, well, findOverlaps:
apoe.i <- findOverlaps(tx.by.gene["348"], my.snps)
apoe.i is an object of class RangesMatching. Note that had we not matched chromosome names, Bioconductor gives us a nice warning that sequence names don’t match. We could look at the slots of apoe.i but output can be seen with matchMatrix:
hits <- matchMatrix(apoe.i)[, "subject"]
hits
[1] 873650 873651 873652 873653 873654 873655 873656 873657 873658 873659
[11] 873660 873661 873662 873663 873664 873665 873666 873667 873668 873669
[21] 873670 873671 873672 873673 873674 873675 873676
So in our subject, we have two hits. Let’s dig them up in our SNP GRanges object:
my.snps[hits]
GRanges with 27 ranges and 2 elementMetadata values:
seqnames ranges strand | rsid genotype
<Rle> <IRanges> <Rle> | <character> <character>
[1] chr19 [50101007, 50101007] * | rs440446 CG
[2] chr19 [50101842, 50101842] * | rs769449 GG
[3] chr19 [50102284, 50102284] * | rs769450 AG
[4] chr19 [50102751, 50102751] * | rs769451 TT
[5] chr19 [50102874, 50102874] * | i5000209 GG
[6] chr19 [50102904, 50102904] * | i5000208 GG
[7] chr19 [50102940, 50102940] * | i5000201 CC
[8] chr19 [50102991, 50102991] * | rs28931576 AA
[9] chr19 [50103697, 50103697] * | rs11542040 CC
... ... ... ... ... ... ...
[19] chr19 [50104077, 50104077] * | i5000212 GG
[20] chr19 [50104118, 50104118] * | i5000210 GG
[21] chr19 [50104129, 50104129] * | i5000213 CC
[22] chr19 [50104154, 50104154] * | i5000207 TT
[23] chr19 [50104177, 50104177] * | i5000219 GG
[24] chr19 [50104180, 50104180] * | i5000218 GG
[25] chr19 [50104198, 50104198] * | i5000206 CC
[26] chr19 [50104268, 50104268] * | i5000204 GG
[27] chr19 [50104333, 50104333] * | rs28931579 AA
Now, we can verify that these SNPs are in the APOE gene using the UCSC Genome Browser (we could actually pull open a browser to this spot from R using rtracklayer, but I’ll save that for another time). Be sure to use hg18/build 36! Note that my genotype information is there.
The ApoE4 allele is rs429358(C) + rs7412(C). The most common allele (ApoE3, or e3/e3) is rs429358(T) + rs7412(C) which is what I have (that’s a relief). There’s a lot of established research that shows homozygous ApoE4 (that is rs429358(C/C) + rs7412(C/C)) leads to substantially higher risk of Alzeheimer’s. According to SNPedia, James Watson requested he not learn his genotype at this locus, and Steven Pinker requested his ApoE data be removed from his PGP10 data.
gwascatWe can use the metadata provided by gwascat to further look for interesting variants in our 23andme data. I would recommend interpreting this data with caution, as summarizing these findings in a single element metadata data frame is hard: there’s definitely lost information.
The gwrngs GRanges object has lots of metadata you should scan through with elementMetadata(gwrngs). The Strongest.SNP.Risk.Allele is useful for seeing what you’re at risk for. First, using the rs ID as a key, let’s join our SNP data with the gwrngs metadata:
gwrngs.emd <- as.data.frame(elementMetadata(gwrngs))
dm <- merge(d, gwrngs.emd, by.x="rsid", by.y="SNPs")
We can search for the risk allele in the 23andme genotype data with R and attach a vector of i.have.risk to the dm data frame:
risk.alleles <- gsub("[^\\-]*-([ATCG?])", "\\1", dm$Strongest.SNP.Risk.Allele)
i.have.risk <- mapply(function(risk, mine) {
risk %in% unlist(strsplit(mine, ""))
}, risk.alleles, dm$genotype)
dm$i.have.risk <- i.have.risk
Now that you have this data frame, you can mine it endlessly. You may want to sort by Risk.Allele.Frequency and whether you have the risk. Because there are quite a few columns in the element metadata, it’s nice to define a quick-summary subset:
my.risk <- dm[dm$i.have.risk, ]
# define some relevant columns
rel.cols <- c(colnames(d), "Disease.Trait", "Risk.Allele.Frequency",
"p.Value", "i.have.risk", "X95..CI..text.")
head(my.risk[order(my.risk$Risk.Allele.Frequency), rel.cols], 1)
rsid chrom position genotype Disease.Trait Risk.Allele.Frequency
2553 rs2315504 chr17 36300407 AC Height 0.01
p.Value i.have.risk X95..CI..text.
2553 8e-06 TRUE [NR] cm increase
This is a rare variant, but the most important next question is, rare in who?
dm[which(dm$rsid == "rs2315504"), "Initial.Sample.Size"]
[1] 8,842 Korean individuals
So this clearly doesn’t mean much to me since I am of European ancestry. We can use grep to find studies that mention “European”:
head(my.risk[grep("European", my.risk$Initial.Sample.Size), rel.cols], 30)
One interesting rs ID that popped up in this list of my data is rs10166942, which is lightly linked to migraines (from which I suffer).
Git Notes [Digital Notebook - Vince Buffalo] 2012-03-11 04:00
These are updated by me periodically. I have tried my best to illustrate common use cases, and the motivation for doing things the “Git” way.
I’ll use this setup scenario frequently. In a suitable scatch repository (i.e. git-sandbox), make a fake remote:
mkdir fake-remote
cd fake-remote
git init --bare
cd ..
Now, clone it, pretending you are two developers:
git clone fake-remote jerry-repo
git clone fake-remote kramer-repo
Let’s assume you’re Jerry and Kramer is another programmer in your group. As Jerry, let’s make some changes:
cd jerry-repo
echo "an example file" > file.txt
git add file.txt
git commit -am "initial import"
git push origin master
cd ..
Now, let’s pretend we’re Kramer and grab that recent commit:
cd kramer-repo
git pull origin master
cd ..
Git remote tracking branches are similar to local branches (i.e. the kind you interact with git checkout -b branch-name and see with git
branch). However, you don’t work on the remote branch directly, you work on a local branch that’s tracking this remote branch. For example, the most common workflow is to track a remote branch, then push your commits to it or pull commits down from it. Even though it’s a “remote” tracking branch, the branch is stored locally (this branch doesn’t disappear if you can’t connected to the remote).
Git remote tracking branches always have the format remote-repo/remote-branch. After cloning a repository, you can set it to track a remote tracking branch with the -u option of git
push, e.g. git push -u origin master. From now on, you can just use git push when on this branch; this branch is tracking origin’s master branch.
git branch shows local branches; to see remote branches use git
branch -r, and to see all branches, use git branch -a.
Remote tracking branches are also what determine what is pulled/pushed when using git pull and git push without a remote repository and refspec (i.e. git push origin master).
If the current branch is new-feature, which tracks origin/new-feature, then any branches checked out from new-feature will also track the remote too, unless --no-track is added.
Recall the above point that remote tracking branches are local, so (unlike Subversion) they function even when you’re not able to connect to the remote. This gives an example of how elegant Git is: being so similar to regular branches, Git remote tracking branches can be merged into local branches. This is precisely what goes on behind the scenes with git pull. Here’s an example. First, let’s set it up such that a developer in your group, Kramer, made some changes, committed them, then pushed them to the remote.
# assuming you're in the right directory
cd kramer-repo
echo "kramer adding gibberish" >> file.txt
git commit -am "I added some gibberish"
git push
cd ..
Now, imagine you (Jerry) have made some commits but want to see what the status of the remote looks like. git remote show can be used to see if the local remote tracking branch is out of date.
vinceb@poisson$ git remote show origin
* remote origin
Fetch URL: /Users/vinceb/Desktop/git-sandbox/fake-remote
Push URL: /Users/vinceb/Desktop/git-sandbox/fake-remote
HEAD branch: master
Remote branch:
master tracked
Local branch configured for 'git pull':
master merges with remote master
Local ref configured for 'git push':
master pushes to master (local out of date)
So we are out of date! We can see these commits before merging them in with git fetch. git fetch updates your remote tracking branch with the new changes, allowing you to diff branches just as you would with two regular branches.
vinceb@poisson$ git diff master origin/master
diff --git a/file.txt b/file.txt
index 4e850ce..34ceb34 100644
--- a/file.txt
+++ b/file.txt
@@ -1 +1,2 @@
an example file
git log origin/master master also works. git log origin/master
^master shows us just the new commits. We could really explore these commits by checkout out the remote tracking branch (but consider this to be “taking a visit”; don’t commit anything). Suppose we did, and we decide we want to merge them with our current branch. For this, we just use git merge: remember remote tracking branches are just branches!
vinceb@poisson$ git branch ## always check what branch you're on!
* master
vinceb@poisson$ git merge origin/master
Updating ee922a9..8c8c240
Fast-forward
file.txt | 1 +
1 files changed, 1 insertions(+), 0 deletions(-)
Note that git pull basically is git fetch && git merge.
The Beauty of Bioconductor [Digital Notebook - Vince Buffalo] 2012-03-08 03:00
In talking with bioinformaticians, biologists, and other researchers, I’ve seen some worrying trends in computation in the sciences. I plan on writing about these extensively in the future, as I believe computation in the sciences will not scale well to face the huge wealth of data coming experiments will provide. This is not due to algorithmic or hardware limitations, but rather to the fact that scientific programmers simply do not have the same standards and practices that the software industry does.

How do we prevent a big genomic data breaking point?
Three events are simultaneously occurring that could endanger the validity of scientific conclusions in the future. First, new technology is providing the average scientist with more data than ever before. Genomics is the prime example of this: the average biologist can now sequence multiple samples simultaneously whereas this would be prohibitively expensive just a few years earlier. As metabolomic and proteomic data are increasingly incorporated into research alongside genomic data, the work done by bioinformaticians will increase significantly. More lines of code to write and more data to process under deadlines will doubtlessly lead to mistakes.
The second contributing factor is that more researchers are writing code and analyzing their own data rather hiring a bioinformatician or statistician. It’s an awesome and commendable occurrence, but sadly academic institutions don’t adequately prepare researchers to code to high standards. Also, in many cases these researchers learn to program by analyzing their own experimental data, rather than example or “toy” data. This makes “silent” mistakes (i.e. those that don’t prompt an error, but lead to incorrect results) impossible to discover as the actual results are not known.
The last contributing factor is that there’s not a strong expectation that coding standards and software engineering practices be upheld in the sciences. There’s a strong cowboy coding culture in scientific programming. In this mindset, the coding is done when the data is processed, not when the data is processed, the code documented, the unit tests passed, the code checked into a repository, etc. The scientific community needs to embrace the idea that proper data analysis takes time: perhaps as long or longer than gathering experimental data.
In future essays I’ll talk more about these issues in depth. This stuff honestly keeps me (and other people I know) awake at night. I worry humanity may face a Thalidomide-like event in the future due to an error in scientific programming.
However, here I want to commend a project that I feel is underutilized in the biology and bioinformatics communities: Bioconductor. It’s worthy of praise as both an example of, and tool to aid in excellency in bioinformatics programming. I’ll focus primarily on its capacity for handling high throughput sequencing data (even though it handles data from other assays very well too).
Currently, many bioinformatics analyses go something like this: experimental data is received, and then a bioinformatician downloads vast amounts of other data relating to the experiment from web resource such as the UCSC Genome Browser, Ensembl, Phytozome, etc. Often this includes genome assemblies, transcript sequences, and annotation data. Then, application code (alignment software, assemblers, SNP finding tools, etc) are downloaded and compiled. These tools are used alongside custom code written that combines downloaded data with the experimental data, and this produces results that are interpreted. Intermediate results may be fed into other online tools and databases like DAVID or Reactome.
However, this is a bad model if one wants the analysis to be reproducible. The common weakness is that web resources can be unstable. It’s then necessary for the researcher to record software and data versions manually. Even if the researcher dutifully complies, outside databases and code repositories may disappear and leave the project unable to be reproduced. Researchers truly invested in conducting reproducible research then have to store data and software versions themselves, which given the scale of genomic data is quite a burden.
Thus, three things currently perplex reproducible research in bioinformatics: the scale of both experimental and other required data prevents easy self-archival, the fast-paced development of bioinformatics tools could lead to differing results across versions, and the overwhelming prevalence of web-based data resources and applications which are not easily reproducible.
Bioconductor has, in my opinion, the best solution to these problems. First, Bioconductor stores past versions of its packages back to the earliest releases. Past experiments can be replicated using the exact version of software that was used for the actual analysis.
Second, Bioconductor stores data as packages. Pre-packaged versioned data is a cornerstone of reproducible research. For example, suppose I am working with human RNA-seq data. This requires transcript annotation data, which could be downloaded from an online resource. To be reproducible, this requires that:
The webhost be up indefinitely.
The URL remain stable and point to the exact same resource.
The user provide not only a URL but the version of data/software downloaded.
That the external resource provider (i.e. database or application developer) actually update their versions accordingly.
For absolute best practice, it’s also necessary to MD5 checksum the data and record this value to maintain any data gathered from the same source is the exact same.
In contrast, consider how I would load human transcript data into R with Bioconductor:
library(TxDb.Hsapiens.UCSC.hg19.knownGene)
The versioning here is done explicitly in the package name: hg19. I could also easily record the state of all my Bioconductor packages and my session with sessionInfo():
> sessionInfo()
R version 2.14.1 (2011-12-22)
Platform: x86_64-apple-darwin9.8.0/x86_64 (64-bit)
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
attached base packages:
[1] stats graphics grDevices utils datasets methods base
loaded via a namespace (and not attached):
[1] annotate_1.32.2 AnnotationDbi_1.17.23 Biobase_2.14.0
[4] BiocGenerics_0.1.12 DBI_0.2-5 DESeq_1.6.1
[7] genefilter_1.36.0 geneplotter_1.32.1 grid_2.14.1
[10] IRanges_1.12.6 RColorBrewer_1.0-5 RSQLite_0.11.1
[13] splines_2.14.1 survival_2.36-12 xtable_1.7-0
Entire genomes are also packaged via the BSgenome package (BS refers here to Biostrings). If the data in packages is not sufficiently recent, the GenomicFeatures package provides a programmatic way of downloading, packaging, and using data from BioMart and UCSC Genome Browser tracks, and provides functions for saving and loading transcriptDb objects from such resources. Recently Duncan Temple Lang and I were speaking about reproducible research, and he said “people adopt best practices when they’re right in front of their face”. Bioconductor’s tools do just that. Furthermore, Bioconductor has strict coding and documentation standards (much stricter than CRAN actually), which ensures user-contributed packages are high quality.
When discussing R and Bioconductor with other researchers, it’s easy to convince them to adopt both for analyzing statistical data - the data that comes in the very final stages of a bioinformatics analysis. It’s usually much more difficult to convince them to consider working with high throughput sequencing data in Bioconductor. Folks complain that it’s (1) not worth it to process sequencing data with Bioconductor tools or (2) it’s not fast enough. I’ll address the second point in a bit; more importantly I want to emphasize that it’s absolutely worth it to process sequencing data in Bioconductor.
In analyzing genomic data, we take very, very, very high dimension data and try to condense it into biologically meaningful conclusions without being misleading or getting something wrong. Every step is about taking dense data and making it understandable: we take sequence reads and try to assemble them into larger contigs and scaffolds, we take cDNA reads and try to map them back to genomes to understand expression, etc. At each step, our tools make heuristic or statistical choices for us. Pipelines woefully ignore these choices because in most cases, after a step is completed, a script jumps to the next step.
When I think about these steps, I try to assess what I think of as “informational leakage” in bioinformatics processing. Each step summarizes something, hopefully in a way without bias or too much noise. Informational leakage is the information that’s lost between steps. Catastrophic information leakage occurs when we lose information that could have indicated whether the data is biased or incorrect. We can hedge the risk of information leakage when we use summary statistics between steps that try to capture this leaked information.
Consider processing RNA-seq reads. The first step is usually quality control, i.e. removing adapter sequences and trimming off poor quality bases. Failing to gather summary statistics before and after each of these steps leads to potentially catastrophic information leakage. Suppose that an experiment with control and treatment groups, sequenced on two different lanes (bad experiment design!). If one lane has systematically lower 3’-end quality than the other, quality trimming software will trim these bases off and lead one experimental group to have much shorter sequences than the other. The mapping rates will differ significantly, as shorter reads may map less uniquely. In the end, our data is completely confounded not only by the lane (and bad experimental design), but by our own tools! If these tools are being run in a pipeline without intermediate summary statistics being gathered, this catastrophic information leakage will go unnoticed.
Loading sequencing data into R and using Bioconductor’s tools earlier allows summary statistics to be gathered earlier and more easily (R is, after all, great for statistics and visualization), which I strongly believe will decrease the risk of catastrophic information leakage in genomics data analysis. This is why I wrote qrqc, which can provide quick summary statistics on sequencing read quality. Used before and after the application of quality tools, qrqc can provide information not only on the state of the data, but also the effect of the tools.

With existing Bioconductor packages, many useful statistics can be gathered on whole reads, BAM mapping results, VCF files, etc.
The complaint that R is slow, and couldn’t possibly be used with sequencing/mapping-level data is unwarranted. In reality, some of Bioconductor’s core packages for working with high throughput sequencing data such as Biostrings and IRanges (the foundation of GenomicRanges) are astoundingly fast because most of their backends are written in C. Biostring actually uses external pointers to C structures and bit patterns to encode biological string data efficiently.
In addition to being fast, they’re also clever. Biostrings implements an abstraction called a “view” on an XString object, which efficiently represents multiple sections of the same string object (such as subsequences of interest). While I wouldn’t write a short read aligner or assembler entirely in R, many bioinformatic tasks are more than sufficiently fast with Bioconductor tools.
In evangelizing Bioconductor, I have two goals. First, I want to spread awareness that it’s the best way to do reproducible bioinformatics that I think exists today. I want more people to use it just because I deeply care about the state of science and reproducibility. Second, I want to build excitement about this project so that more people will contribute. I believe that far too many high quality bioinformatics tools are written outside of Bioconductor. Packaging bioinformatics tools in Bioconductor forces the developer to adopt strict standards, write clear documentation, and open up a program to a large, active user base. Any results from a package’s methods can then easily be evaluated using R, CRAN, or Bioconductor’s existing tools.
I also believe that large programs (like BLAST, and maybe assemblers) should provide better interfaces to R, to prevent information leakage in analysis. I’m willing to bet that a large majority of bioinformatics tools could be outputting more statistics than they currently do that could be valuable in assessing their functionality. R interfaces to these bioinformatics tools will drastically make it easier for biologists and bioinformaticians to prevent information leakage.
Thoughts on Julia and R [Digital Notebook - Vince Buffalo] 2012-03-07 03:00
Julia is an exciting new technical computing language. It’s still in its infancy, but it’s fast (see below), and already does a lot.

There’s been some excitement on Twitter about Julia. Excitement combined with open source often yields development, which then leads to further excitement, until a mature open source project arises. One of Julia’s explicit goals is to challenge other statistical computing environments, including R.
R is, without a doubt, changing the world. It’s being used by industry giants like Facebook and Google, while also providing academic researchers in statistics, biology, psychology, and countless other fields with not only a free and open source statistical environment, but a huge number of user-contributed package through CRAN. Now methods papers in many fields are often accompanied by CRAN or Bioconductor packages. It’s also a brilliant platform for reproducible, open research, as Bioconductor beautifully illustrates with packaged and version-controlled genomes, microarray probesets, etc.
However, R is suffering from growing pains. For example, there are now 64-bit versions of R, however, vector indexing is still limited by R_len_t (see definition in src/include/Rinternals.h):
/* type for length of vectors etc */
typedef int R_len_t; /* will be long later, LONG64 or ssize_t on Win64 */
#define R_LEN_T_MAX INT_MAX
It appears that one can simply change this to a long and recompile to increase the longest possible addressable vector, but no. Take a look at R_euclidean in library/stats/src/distance.c for an example why: almost all variables for iterating over elements in vectors are defined as integers and don’t use this type. One would have to read through every function, and every line of code to fix this.
R_len_t is just one example. Another issue is that R has been slow to adopt new compiler technologies (i.e. JIT, optional type indications, etc). R almost always gains speed from pushing stuff to C (the recent bytecode compiler is an exception). This isn’t a problem, but it’s a huge limitation to require developers to not only know R, but also C, and also how to interface the two. More modern languages (Java, as well as Python and Julia come to mind) spend more time tracking compiler technology developments and implementing them than R core does (again, Luke Tierney and the bytecode compiler are exceptions). It’s still sometimes efficient to use C with these languages (consider Cython), but developers in these language aren’t cracking open Kernighan and Ritchie everytime they need to have a for loop do something quickly.
Another gripe I have is that R language development is somewhat closed. Despite a quickly expanding user base, the number of R core contributors is not increasing. I find it hard to believe this is due to lack of interest. It seems much more likely this is due to institutional reasons that need to be changed. The nice thing about language development that it’s really hard, so opening up R to more contributors won’t likely flood the existing core with bad ideas and patches. Personally I would dedicate much more time profiling, reading the source, and working on the R language if it were more open.
The last gripe I have is that R is fragmented. Consider Python:
import re
re.search(r'R-([\d]+).([\d]+)', "R-2.15").groups()
Now, consider R:
gsub("R-([\\d]+)\\.([\\d]+)", "\\1", "R-2.15")
# or
library(stringr)
str_match("R-2.15", "R-([0-9]+)\\.([0-9]+)")
Now, Python also has PyPI’s re2, but most developers are using re. The motivation behind stringr is that R’s currently family of string processing functions are horribly inconsistent:
# (my ... to avoid writing all parameters)
grep(pattern, x, ...)
regexpr(pattern, text, ...)
gsub(pattern, replacement, x, ...)
strsplit(x, split, ...)
But rather than deprecate these and move forward, we now have two sets of string processing functions. Both are being used. I’m not saying Hadley Wickham is to blame here; quite the contrary, he’s trying to fix a very annoying problem in the language and should be commended. I think the community needs to be more open; for example, before writing a package that processes strings, let’s discuss an implementation plan, deprecating old functions, etc. If not, in the future R will be highly fragmented, and end up with five different object orientation systems… oh, wait.
Contributors to Julia are optimistic they can challenge R based on a solid foundation of JIT compiling, parallelism, and nice language semantics. I salute this optimism, but I think we need to realistically consider what it would take to “challenge” R.
First, we would need to build an equal statistical computing environment. Consider moving all of stats, MASS, graphics, grid, etc to Julia. Is Julia sufficiently faster than R will be in the time it takes to port these base packages? Remember, R is a moving target; despite my few earlier gripes, R will evolve and get faster. Now, consider adding the extremely popular CRAN packages like ggplot2 and lattice to Julia. In the time it takes to port these, is Julia still sufficiently faster than R will be?
Suppose it is still faster than R. What about after we port the rest of CRAN, and all of Bioconductor to Julia? My point isn’t say that it’s unimaginable that Julia will surpass R. It’s that developers should really dissect what makes a successful language successful before they try to challenge it. I don’t have a horse in this race; I would love to see Julia surpass R. But if all developers want is a fast environment to analyze large data sets using a wealth of methods and libraries, it may be a lot easier to make R faster than to develop a new fast language and hope/wait/beg the community to move over.
Some Ramblings on Machine Learning in Science [Digital Notebook - Vince Buffalo] 2012-03-03 03:00
In between refactoring some qrqc code this morning and looking at RNA-seq data, I grabbed some cold brew coffee and caught up on some missed tweets. Admittedly, my brain glosses over most tweets, but this tweet from Drew Conway had the right mix of keywords to actually make me click and read the link:
The data science debate: domain expertise or machine learning? by @medriscoll http://bit.ly/zr17Z2
I don’t mean for this title to be inflammatory, but I do believe this debate is a bit unbelievable. Machine learning is magical; I imagine that everyone that has studied it goes through a hype cycle-like set of epiphanies. This is my hype cycle story, and why I believe machine learners need to calm down, collaborate with domain experts, and together tackle hard problems.
MONIAC: social science machine learning?
Biologists, it’s true: I’m not one of you. I’m a transplant from the social sciences. Specifically, from political science and economics (with statistics too), where my interests lie in methodology and comparative politics.
In the social sciences, the dimensionality of most problems is small enough that data mining is (at least in my experience) frowned upon. A lot of political data is collected by hand, often by undergraduates toiling away for meager pay as they try to understand some cryptic event coding protocol. There are some very large p data sets: The Political Instability Task Force’s data set is something I’ll keep mentioning. Mining this data with algorithms looking for interesting relationships was exactly how I was taught not to do political science.
I recall one story of a candidate giving a job talk mentioning he used forward step-wise regression to find interesting variables (in a presumably small p data set) and three people immediately stood up and left. I was proud to be knowledgeable of, but avoid statistical learning techniques. Political science had flirted with neural networks to understand massive state failure data sets, but my endless gripe was there these were predictive, not causal models. The latter required some a priori testable theory, often derived from an intimate knowledge of political crisis in a variety of countries. Just as I thought biologists knew c. elegans or s. cerevisiae well enough to form interesting experiment ideas, political scientists knew many political crises well enough to form theories and test them on a larger set of data in a quantitatively rigorous fashion. I also believed that predictive models of state failure may predict recorded (even when out-sample!) state failures well, but a model backed in a good theory that fits existing data slightly less well could predict unseen cases even better (Bruce Bueno de Mesquita has an entire wonderful book about game theory being such a model).
When I made the jump to analyzing gene expression data, I was initially astounded at how many algorithms were thrown at it. I had this vision of the hard sciences having randomization and experimentation at their disposal to lead to the purest causal findings. Looking for any differences in 30,000 genes’ expression values and then forming hypotheses after seemed backwards. Microarrays shocked biology with what they revealed about cancer and the cell, but they probably shocked the methods of experimental biology more. If your average biologist had a tenuous knowledge of p-values to begin with, now microarrays analysts were throwing around false discovery rates, empirical Bayesian techniques, Storey’s q-value, etc.
However, as I analyzed more and more sets of data, the initial reluctance I had about employing machine learning algorithms disappeared. In hype cycle terms, I was climbing the peak of inflated expectations. A quote from Michael E. Driscoll’s article captures this excitement:
Claudia Perlich, a three-time winner of the KDD Nuggets competition, stood up and shared how she had won contests in domains as varied as “breast cancer, movie prediction, and sales performance - and I can tell you I knew next to nothing about those things when I started.”
This optimism is abundant, and not entirely without justification. Coming from a non-biological background yet thoroughly understanding machine learning provides an immensely satisfying feeling of understanding of the cell. Employing all sorts of machine learning techniques, I could find “biologically interesting” genes in data sets and help biologists understand the cell.
The Hype Cycle’s lowest stage is the “trough of disillusionment”. Machine learning in biology certainly hasn’t had its trough (and I don’t think it will), but it is priming up to have its “slope of enlightenment” and “plateau of productivity”. There will be future machine learning hype cycles in biology, especially as multiple heterogeneous data sets need to be simultaneously mined to understand the cell with the systems approach.
My personal dip didn’t happen because machine learning left me with a particularly terrible result - it occurred because (1) because of an interaction I had with an experimental biologist and (2) I realized how wonderfully complex the cell is.
The first interaction I had was with a graduate student friend of mine. We were discussing an interesting finding the Korf Lab made: that some introns lead to increased expression (paper here). Introns traditionally haven’t had the same attention as promoters of enhancers in regulating gene expression. A member of the Korf lab had previously mentioned intron-mediated expression in passing to me, and I immediately started imagining what ways I could look for such an effect in silico. As I understood it, in silico was how it was first discovered, further increasing my admiration of algorithms applied to biology. When my friend mentioned it again, the first thing she said was, “well, we just need to take that intron and put it in something”.
I immediately agreed, but I realized something: I hadn’t thought of that simple step the first time I thought about intron-mediated expression. Machine learning can bring so much wealth in finding interesting relationships that my mind had glossed over the most important question in science: whether these relationships were spurious or causal. This is why my training in the social sciences was rigidly anti-machine learning: it’s far too easy to let our thought processes about understanding a complex system be biased by some spurious relationships machine learning and predictive models can quickly give us.
The epiphany was gradual (and still occurring): the cell is wonderfully complex, or as my mind puts it “fucking awesomely complex”. Machine learning applied to gene expression data gives valuable insights into a complex system, but it’s really a messy snapshot. I think we’ll look at current pristine RNA-seq experiments in twenty years and we’ll realize they’re giving us an image into cellular activity that is akin to a photograph from a cheap Soviet-era camera.
Measuring gene expression from many cells glosses over interesting variation in each cell; this is certainly not a new complaint. However, even a single cell image is messy: mRNAs that make their way into gene expression values may have never been exported from the nucleus, they could have been degraded by the cell, silenced, undergone post translational modification, etc. What’s astounding is that these systems are not just complex, but are amazingly accurate. Cellular data is messy, but the cell certainly isn’t. Development is a prime example of how tightly regulated these processes are. It’s up to us to understand these tightly regulated systems with the messy images scientific data gives us. Machine learning is a necessary, but not sufficient tool to help us understand the cell.
As an example, genes interact in groups, and many algorithms can gloss over this detail. If an algorithm tries to find a sparse set of genes that are biologically interesting to the problem at hand, it may be indifferent to which it includes from a set of co-expressed genes (consider the lasso against the elastic net here). If a biologist reviews these findings, they could easily miss something vastly important based on machine learning’s indifference.
These epiphanies are now what guides my path through biology and machine learning. I still love and am infatuated with machine learning (although, I much prefer the phrase statistical learning). However, if we wish to understand a complex system, we need to take the approach that modern biology does: leverage machine learning with a priori biological expert knowledge to bootstrap findings. We need to design experiments that also harness the power of machine learning to help us understand, and not just predict the behavior of complex systems. Applied machine learners need to realize the power of experimental data. Chances are if you’re finding everything you think is out there with just machine learning, you’re making a mistake or your problem is too simple.
Elucidating k-mer Contamination with Kullback-Leibler Divergence [Digital Notebook - Vince Buffalo] 2012-03-01 03:00
Recently a coworker showed me a FASTQ file from an Illumina HiSeq run (which will be packaged in the new release of my Bioconductor package qrqc) that was severely contaminated. Below is the file in less with a string highlighted:

Holy contamination, Batman! There are a few approaches to handling this level of contamination. The program tagdust will match contaminated reads and remove them. My program Scythe is being changed so that it can match adapter contaminants further in the read using its Bayesian model. Both programs require a priori knowledge of possible contamiant sequences - what if this is a novel sequence contaminant? In this case, AAGCAGTGGTATCAACGCAGAGT appears to be a PCR primer related to DSN normalization that may not have made it into our adapter files.
k-mer approaches are a nice way of searching for such contaminants. I am currently adding this feature to qrqc; you can follow development on the kmer branch on Github but this branch may merge into master and disappear.
The C functions I’ve written use Heng Li’s khash.h library to quickly hash k-mer sequences with their positions in the read. The end result is a data frame in R of k-mer sequence, position, and counts in that position.
Looking at raw k-mer counts is somewhat useful, but I’ve been exploring some information theoretical approaches to analyzing this data. One useful graphic is entropy of k-mers by position:

These are 6-mers, so there are 4,096 possible k-mers (excluding N). If the k-mer distribution were uniform, 12 bits would be needed to encode each k-mer. This graph illustrates that even at the most random 3’-end of the read, only about 6.5 bits are needed. In the first 20 bases, the distribution of k-mers is so skewed that less the Shannon entropy is less than four bits.
It makes sense biologically that the k-mers don’t have uniform frequency. In the case of read contaminants, the enrichment by position against an empirical k-mer distribution may be as interesting as total k-mer enrichment against a random distribution model.
To assess this, some beta qrqc code pools k-mer counts across position to find an empirical k-mer distribution. Then, the k-mer distribution per position is compared to the pooled distribution using the Kullback-Leibler divergence. K-L divergence is only defined when both distributions sum to 1, the sample spaces are the same, and if q(i) > 0 for any i such that p(i)> 0.
In essence, the K-L divergence is measuring the average number of bits needed to encode data from P with a code based on the distribution of Q. In the k-mer case, Q is the empirical distribution of k-mers irrespective of k-mer position and P is the position-specific distribution of k-mers. Thus, an enrichment of k-mers at a particular position would lead to more divergence.
A nice feature of ggplot2 is the stacking of the “bar” geom. Since K-L divergences are sums, stacking and setting fill color by k-mer (the terms of the sum) gives us a sense of the total divergence and each k-mer’s effect on the total. There are too many k-mers to plot, so I have some procedures that find a nice subset. Because this is a subset, the K-L total (indicated by bar height) is wrong, but the graphical interpretation is easier. Now, the enrichment by position is clear:

This messy dataset has repeat primer contamination. Note that because we’re plotting a subset of k-mers, there is negative total K-L (not mathematically possible) because we’re leaving out terms in the sum, but the meaning still comes through. Also note that there is k-mer nesting: The first wide peak begins with k-mer TATCAA, then ATCAAC, then TCAACG, etc. This indicates that we could adjust k and find the entire repeated k-mer.
I’ve added faceting of multiple SequenceSummary objects’ KL/k-mer diagnostics. Combined with a random data file, this really illustrated contamination:
This is still in development; follow the code on Github and feel free to contact me and make suggestions.
Articles of Note, by Topic [Digital Notebook - Vince Buffalo] 2012-02-23 03:00
Please developers, don't be dicks. [Digital Notebook - Vince Buffalo] 2012-02-21 03:00
As the author of a few open source tools, I’ve had my fair share of users seeking help. Emails range from the very useful (bug reports, patches, etc) to the annoying (“can you help guide me through this process”). But never once (that I can remember) have I been a dick (and yes, I’ve wanted to be). It will be tricky to write this without sounding self-righteous, but I hope to make the case that open source developers shouldn’t be dicks in all cases.
The first reason to never be a dick is that We’ve All Been There (I’m going to give this the acronym WABT). Even the most voracious and diligent manual readers can suffer from the XY problem. A user comes asking how to do Y, which they think is the solution to X. However, it’s a bad solution to X and they don’t know this. These situations will always lead to frustration: users waste time explaining Y and helpers waste time explaining how to do Y to realize the user wanted X. But this is not the user’s fault; it just takes programming practice to realize Y is not the correct way to do X.
We’ve all had these problems in our early stages as developers. Being a dick in these cases will not help the user grok anything. They’re already frustrated - that’s why they’re asking for your help. Being a dick will cause them to get more frustrated and really not grasp anything. They’re not going to have an “ah ha!” moment when they’re too busy trying to come up with a witty response to your burn on IRC.
Please Check The Manual (PCTM) has the same number of letters as Read The Fucking Manual (RTFM). I strongly believe it takes more energy for a developer to be a dick than to be nice. We’ve all had dumb questions that disrupt our workflow, make us angry, etc. But being a dick back does not discourage this behavior. Write some boilerplate text for responding to users’ questions. Make this a FAQ. Then respond, PCTM (Please Check the Manual) and send them the link. If they get needy, tell them open source software doesn’t come with a warranty.
Someone was once quite rude to me via email (I’ll name him Tom). I had voiced some frustrations with software Tom wrote and he attacked me for these public comments. Now, as an aside, there’s a lot of shitty software out there, and signals about software quality (even noisy signals) are very valuable. Tom on one hand attacked me for saying something negative about his software, and on the other hand asked me to help fix it, emphasizing it was open source software. I agree with this sentiment 100%, however the email was clearly very angry.
I told another developer who I’ll call Jerry about the encounter, and he laughed. Apparently, Tom nagged Jerry about portability issues of Jerry’s software years ago. This is evidence of my first point, WABT. It also shows that developers remember interactions with other developers really well. Since then, I’ve also heard other programmers complaining about interactions with Tom. This is all too bad, as Tom is probably very nice in person and certainly a good programmer.
OSS has seen an explosion in recent years. Biologsts, ecologists, and social scientists that never thought they’d write code are using R to analyze data. Folks frustrated by Windows are installing Ubuntu and asking for help. In the early days of the OSS, usenet, and IRC, it was an acceptable norm to be a dick. Now, it’s not.
OSS benefits from a large user base, but it will have growing pains. Being a dick does not alleviate these pains, it makes them worse. Let’s go back to my story about Tom.
In the second half of Tom’s email (after attacking me), he asked me to help him fix his software. Now, collaboration can be difficult; code style clashes, merges fail, frustration is common. In a small project, you’re really in bed with your collaborators. Now that Tom has sent me the signal he’s nasty in correspondences, do you think I’ll work on this project with him? Hell no. I’d rather fork, fix the problem and encourage others to use my software. Of course this is bad for OSS; consider this passage from Eric S. Raymond’s Jargon File:
Forking is considered a Bad Thing - not merely because it implies a lot of wasted effort in the future, but because forks tend to be accompanied by a great deal of strife and acrimony between the successor groups over issues of legitimacy, succession, and design direction. There is serious social pressure against forking.
Tom’s actions guarantee I will avoid working on his projects at all costs. The two other developers, and anyone else we’ve told will too. In the end, the software loses.
Some abrasive programmers are really gifted. Erik Naggum is regarded as the first Usenet flamer. Theo de Raadt forked NetBSD into what became OpenBSD partially because of issues with other developers. Richard Stallman gave an AMA on reddit a year ago and the most popular question (since deleted) was about a young GNU-lover that was nervous about asking RMS a question and accidentally referred to it as “Linux”, not “GNU/Linux” and RMS ripped him in half.
Now, all of these developers have been dickish and are well-known because they are gifted visionaries. I’m not sure why, but other developers admire this dickishness. But don’t idolize their dickishness, idolize their skill. There are also overwhelmingly nice programmers: John McCarthy, Donald Knuth, and Alan Turing to name a few. Admire their skill and their personality.
There’s been an explosion of open source software utilization in the sciences. My field, bioinformatics, provides an interesting case study. There are bioinformaticians like myself that write software. Users are divided into other programmer types (other bioinformaticians) and biologists (on average, less knowledgeable of programming). All else equal, biologists and bioinformaticians prefer free, open source software to costly proprietary software.
For these reasons, being helpful and nice to scientific users is really important. For biologists, choosing tools is about getting analysis done quickly and easily. Rude bioinformaticians will quickly increase the cost of using OSS tools, which is already high for many biologists who aren’t experienced with Unix tools and programming. Consequently, science could become less open, something neither group wants.
Arduino Notes [Digital Notebook - Vince Buffalo] 2012-02-21 03:00
The following are some notes/links I’ve come across when working with my Arduino Uno.
Ultimately I want to develop through Emacs, not the Arduino IDE. Doing this well involves (1) a good mode for Emacs, (2) interfacing with avr-gcc, and (3) a makefile or script that uploads the binary to the Arduino.
Hello, static blogging [Digital Notebook - Vince Buffalo] 2012-02-20 03:00
I spend hours each week in Emacs, and I’ve spent ages customizing it. With Jekll, Markdown, and Blueprint I quickly built this site in my favorite editor. I can spend time writing content, not fighting a browser WYSIWYG editor.
Also, I want this site to function as a notebook. Most blogging systems lead to a post-and-forget mentality. Revision control makes, well, revisions easier.
Plaintext is powerful (see org-mode if you don’t believe me). If I want to search all my posts using regular expressions, I can with grep. How could this be done if I was using WordPress or any blogging system that used database backend?
Jekyll plays well with Pygments, a Python syntax highlighter. This is import because I want to share code.
# Jekyll is code friendly
x <- seq(-1, 3, 0.1)
y <- sin(x) + rnorm(length(x), 0, 0.3)
plot(x, y)
This was my final worry - org-mode is a bit “heavy” for publishing quick notes (although entirely necessary for other things), but it has excellent LaTeX integration. I would be very disappointed if I couldn’t easily incorporate LaTeX into Jekyll. Luckily, I can! The default Maruku Markdown interpreter just needs to have LaTeX support turned on (see below)… and wahla! I can include math:

There’s a myriad of Ruby tools and plugins for Jekyll - it’s all a bit intimidating. Here are a few notes on my configuration. My _config.yml file is definitely important.
I am using the default Markdown parser, Maruku. Maruku has formulae support, but it experimental and not on my default. Formulae are very important to my notebook, so getting them to work is paramount. Here’s how I did it:
1. Add the Maruku configurations below. Note the trailing slashes - these are important (and a sign that LaTeX support is still a bit rough.)
maruku:
use_tex: true
use_divs: true
png_engine: blahtex
png_dir: images/latex/
png_url: /images/latex/
2. Install Xerces-C. On a Mac with MacPorts, use sudo port install
xercesc. This is a requirement for blahtexml, which Maruku uses.
3. Install blahtexml. Get the source at http://gva.noekeon.org/blahtexml and on a Mac, use make
blahtex-mac and put this binary in /usr/local/bin/ or some other place in your $PATH.
Blueprint CSS has a class highlight in screen.css that clashes with Pygment (giving code a hideous yellow background). I just renamed the class highlight-alt in screen.css and everything looks great now.
Github’s Jekyll engine apparently runs with jekyll --pygments --safe which disables plugins. LaTeX to PNG rendering is built into Maruku, not a plugin, yet builds still fail on Github (even though they are fine with these options locally). I also host this site at http://vincebuffalo.org, so there’s not a huge problem here.
Updated comparison of variant detection methods: Ensemble, FreeBayes and minimal BAM preparation pipelines [Blue Collar Bioinformatics] 2013-10-21 06:35
I previously discussed our approach for evaluating variant detection methods using a highly confident set of reference calls provided by NIST’s Genome in a Bottle consortium for the NA12878 human HapMap genome, In this post, I’ll update those conclusions based on recent improvements in GATK and FreeBayes.
The comparisons use bcbio-nextgen, an automated open-source pipeline for variant calling and evaluation that identifies concordant and discordant variants with the XPrize validation protocol. By having an automated validation workflow attached to a regularly updated, community developed, variant calling pipeline, we can actively track progress of variant callers and provide updates as algorithms improve.
Since the initial post, There have been two new GATK releases of UnifiedGenotyper and HaplotypeCaller, as well as multiple improvements to FreeBayes. Additionally we’ve enchanced our ensemble calling method, which combines inputs from multiple callers into a single final set of calls, to better handle comparisons with inputs from three callers.
The goal of this post is to re-evaluate these variant detection approaches and provide an updated set of recommendations:
Avoiding the post-alignment BAM recalibration and realignment steps allows us to save significant time and pipeline complexity. Combined with the improvements in FreeBayes, this enables a variant calling pipeline that can be freely used for academic, clinical and commercial work with equal quality variant calls compared to current GATK best-practice approaches.
We called variants on a NA12878 exome dataset from EdgeBio’s clinical pipeline and assessed them against the NIST’s Genome in a Bottle reference material. Full instructions for replicating the analysis and installing the pipeline are available from the bcbio-nextgen documentation site. Following alignment with bwa-mem (0.7.5a), we post-processed the BAM files with two methods:
We then called variants with three general purpose callers:
Finally, we evaluated the calls from each combination of variant caller and BAM post-alignment preparation method using the bcbio.variation framework. This provides a summary identifying concordant and discordant variants, separating SNPs and indels since they have different error profiles. Additionally it classifies discordant variants. where the reference material and evaluation variants differ, into three categories:
Using this framework, we compared the 3 variant callers and combined ensemble method:
In addition to calling sensitivity and specificity, an additional factor to consider is the required processing time. Rough benchmarks on family-based calling of whole genome sequencing data indicate that HaplotypeCaller is roughly 7x slower than UnifiedGenotyper and FreeBayes is 2x slower. On multiple 30x whole genome samples, our experience is that calling can range from 10 hours for GATK UnifiedGenotyper to 70 hours for HaplotypeCallers. Ensemble calling requires running all three callers plus combining into a final call set, and for family-based whole genome samples can add another 100 hours of processing time. These estimates fluctuate greatly depending on the compute infrastructure and presence of longer difficult genomic regions with deeper coverage, but give some estimates of timing considerations.
Given the improved accuracy of local realignment haplotype-based callers like FreeBayes and HaplotypeCaller, we explored the accuracy cost of removing the post-alignment BAM processing steps. The recommended GATK best-practice is to follow up alignment with identification of duplicate reads, followed by base quality score recalibration and realignment around indels. Based on whole genome benchmarking work, these steps can take as long as the initial alignment and scale poorly due to the high IO costs of manipulating large BAM files. For multiple 30x whole genome samples running on 16 cores per sample, this can account for 12 to 16 hours of processing time.
To compare the quality impact of avoiding recalibration and realignment, we performed the identical alignment and variant calling steps as above, but did minimal post-alignment BAM preparation. Following alignment, the only step performed was deduplication using samtools rmdup. Unlike Picard MarkDuplicates, samtools rmdup handles piped streaming input to avoid IO penalties. This is at the cost of not handling some edge cases. Longer term, we’d like to explore biobambam’s markduplicates2, which implements a more efficient streaming version of the Picard MarkDuplicates algorithm.
Suprisingly, skipping base recalibration and indel realignment had almost no impact on the quality of resulting variant calls:
While GATK UnifiedGenotyper suffers during indel calling without recalibration and realignment, both HaplotypeCaller and FreeBayes perform as good or better without these steps. This allows us to save on processing time and complexity without sacrificing call quality when using a haplotype aware realigning caller.
Taken together, the improvements in FreeBayes and ability to avoid post-alignment BAM processing allow use of a commercially unrestricted GATK-free pipeline with equal quality to current GATK best practices. Adding in GATK’s two callers plus our ensemble combining method provides the most accurate overall calls, at the cost of additional processing time.
It’s also important to consider potential drawbacks of this analysis as we continue to design future evaluations. The comparison is in exome regions for single sample variant calling. In future work it would be helpful to have population or family based inputs. We’d also like to prepare test datasets that focus specifically on evaluating the quality of calls in more difficult repetitive regions within the whole genome. Using populations or whole genomes would also allow use of GATK’s Variant Quality Score Recalibration as part of the pipeline, which could provide improved filtering compared to the hard-filtering approach used here.
Another consideration is that the reference callset prepared by the Genome in a Bottle consortium makes extensive use of GATK tools during preparation. Evaluation of the reference materials with FreeBayes and other callers can help reduce potential GATK-specific biases when continuing to develop reliable reference materials.
All of these pipelines are freely available, open-source, community developed projects and we welcome feedback and contributors. By integrating validation into a scalable analysis pipeline, we hope to build a community interested in widely accessible calling pipelines coupled with well-evaluated reference datasets and methods.
Summary from Bioinformatics Open Science Codefest 2013: Tools, infrastructure, standards and visualization [Blue Collar Bioinformatics] 2013-07-18 18:26
The 2013 Bioinformatics Open Source Conference (BOSC) starts tomorrow in Berlin, Germany. It’s a yearly conference devoted to community-based software development projects supporting biological research. Members of the Open Bioinformatics Foundation discuss implementations and approaches to better provide interoperable and reusable software, libraries and pipelines.
For the past five years, a two day Codefest and hackathon preceded the conference. This gives programmers time to work face-to-face, sharing approaches and discovering connections between projects. This year, the the Department of Biology, Humboldt-Universität zu Berlin kindly hosted Codefest 2013. Thanks to the organizers and attendees, we finished projects ranging from tool development, infrastructure integration, standards development and visualization. There are photos of the Codefest in progress and a detailed writeup of projects.
Below we summarize the accomplishments from the two days. We welcome feedback on the topics covered and hope that by sharing our work we can encourage more programmers to become part of the open science bioinformatics community. Actively working to build well-tested, community-developed, interoperable tools is how we solve increasingly difficult research questions ranging from human health to plant breeding to microbial community function. The progress made in two days illuminates the effectiveness of open collaborative science.
Toshiaki Katayama, Raoul Bonnal, Eri Kibukawa, Joachim Baran, Dan MacLean, Fernando Izquierdo-Carrasco, Spencer Bliven
During the Codefest, we tested and documented our port of the BaseSpace Python SDK to Ruby. Ruby/Biogem developers can now easily utilize next-generation sequencing code within the Illumina’s BaseSpace framework. For non-Ruby programers, we found that it can be a burden to create new Web app from scratch on top of your NGS program. So we started new project to provide a Web-app scaffold for BaseSpace. We have already implemented the basic portion but will need some more time before releasing the BioBaseSpace application. The BaseSpace Ruby SDK was officially released: for more information, see Joachim’s blog post, the official announcement from the BioRuby team and the annoucement from Illumina.
Torsten Seemann, Tim Booth
For the last 8 years RNAmmer has been the standard tool for predicting ribosomal RNA features in genomes, because it is reasonably fast, accurate, and works on bacteria and eukaryotes. Its drawbacks are that it relies on small, older databases; requires an older conflicting version of HMMER; and has restrictive licence terms. To resolve these issues we have implemented a new rRNA predictor which uses the new “nmmer” tool from HMMER 3.1 for searching DNA profiles against DNA sequence. We used the Silva and GreenGenes seed alignments for the 5S, 23S and 16S genes to build the profile models from. Barrnap is a small Perl script which takes FASTA as input, and outputs the rRNA feature predictions in GFF3 format. It will be packaged in Bio-Linux and replace RNAmmer in the Prokka bacterial annotation system.
Spencer Bliven, Andreas Prlic, Markus Gumbel
Both Java and Scala run on the Java Virtual Machine. As such, it makes sense to coordinate and document the various Bio* projects which run on the JVM and therefor can interoperate to some degree. We are able to successfully reference BioJava functions from Scala code and ScaBio functions from Java code. The ease of this process means that users can easily use both libraries from whichever language is more suited for their biological problem.
Peter Cock, Konstantin Tretyakov, Bin Zhang
The Biopython team worked on training new users at Codefest and exploring integration of Biopython with other Python molecular visualization toolkits like PyMol. Infrastructure development involved testing and debugging on multiple systems, including identifying and fixing Windows and PyPy problems. We also identified areas where we can make it easier to contribute to Biopython: specifically easing the process to report and fix bugs by moving to integrated GitHub issue tracking and working to support Biopython-associated projects with easy installation tools.
Tim Booth
I spent several hours revisiting previous work on the Galaxy package for Bio-Linux and made significant progress towards it being something that can go into Debian-proper. Results will be committed to Deb-Med public SVN and patches will be forwarded to the Galaxy dev mailing list.
Herve Menager, Bertrand Neron, Jackie Quinn, Stian Soiland-Reyes, Matus Kalas, Steffen Moller
The goal of this group was to investigate and implement solutions to use ontologies to help people find and use the programs and data they need for their work, and to help automate the integration of tools or data resources into workflows or workbenches. We also wanted to identify useful provenance metadata, to store in a rigorous way the conditions and configuration of analysis steps run by users. This improves transparency, reproducibility, and reliability of the scientific results.
We worked toward inclusion of the EDAM onotology as part of the Mobyle system’s built-in type and classification mechanisms. We created a user case by identify workflows in Mobyle and mapped the descriptions unto EDAM classification to allow mapping between the types. We also investigated the possibilities opened by projects such as PROV to standardize the provenance information stored by systems such as Mobyle. We added a prototype functionality to the development version of Mobyle that dynamically generates this provenance information in a JSON-based format.
David Powell, Thomas Down, Skyler Brungardt, Alex Kalderimis
We worked on integrating two visualization tools: the Dalliance genome browser and the DGE-Vis RNA-seq explorer. We now have a proof-of-concept tool that makes it possible to visualise RNA-seq analysis while browsing the genome. This inspired a JavaScript scheduler that is able to schedule slow animation updates when the JavaScript engine is not busy, allowing smoother animations and more accurate windows. Finally, we added a JBrowse-compatible JSON backend for Dalliance for integration with Intermine.
Enis Afgan, John Chilton, Brad Chapman
Valentine Svensson, Guillermo Carrasco, Roman Valls, Per Unneberg
We worked to extend the Ipython parallel cluster framework to support additional schedulers, specifically implementing SLURM support to supplement existing SGE, LSF, Torque and Condor schedulers. We plan to extend this to allow generalized use of the DRMAA connector, ultimately port such generalization into ipython so that python scientific computations can be executed efficiently across different clusters implementation. Both Roman and Guillermo blogged detailed documentation of the work in progress.
We also worked to build a tool that helps provide run time estimations for bioinformatcs jobs (e.g. “how long should aligning 40 million reads against hg19 with BWA take if I use 8 cores?”). We plan to collaborate on longer term development of this with the Genome Comparison of Analytic Testing team.
Clare Sloggett, Bernie Pope
We worked on code cleanup, documentation and test data for a reusable pipeline to handle variant calling and annotation, using Rubra built on the Ruffus framework. It handles BWA alignment, GATK alignment cleaning and variant calling and ENSEMBL annotation. To make these pipelines easier to run, we worked on integrating them into the GVF flavor in CloudBioLinux.
Scaling variant detection pipelines for whole genome sequencing analysis [Blue Collar Bioinformatics] 2013-05-22 06:50
Moving from exome to whole genome sequencing introduces a myriad of scaling and informatics challenges. In addition to the biological component of correctly identifying biological variation, it’s equally important to be able to handle the informatics complexities that come with scaling up to whole genomes.
At Harvard School of Public Health, we are processing an increasing number of whole genome samples and the goal of this post is to share experiences scaling the bcbio-nextgen pipeline to handle the associated increase in file sizes and computational requirements. We’ll provide an overview of the pipeline architecture in bcbio-nextgen and detail the four areas we found most useful to overcome processing bottlenecks:
This overview isn’t meant as a prescription, but rather as a description of experiences so far. The work is a collaboration between the HSPH Bioinformatics Core, the research computing team at Harvard FAS and Dell Research. We welcome suggestions and thoughts from others working on these problems.
The bcbio-nextgen pipeline runs in parallel on single multicore machines or distributed on job scheduler managed clusters like LSF, SGE, and TORQUE. The IPython parallel framework manages the set up of parallel engines and handling communication between them. These abstractions allow the same pipeline to scale from a single processor to hundreds of node on a cluster.
The high level diagram of the analysis pipeline shows the major steps in the process. For whole genome samples we start with large 100Gb+ files of reads in FASTQ or BAM format and perform alignment, post-alignment processing, variant calling and variant post processing. These steps involve numerous externally developed software tools with different processing and memory requirements.
A major change in the pipeline was supporting creation of heterogeneous processing environments targeted for specific programs. This moves away from our previous architecture, which attempted to flatten processing and utilize single cores throughout. Due to algorithm restrictions, some software requires the entire set of reads for analysis. For instance, GATK’s base quality recalibrator uses the entire set of aligned reads to accurately calculate inputs for read recalibration. Other software operates more efficiently on entire files: the alignment step scales better by running using multiple cores on a single machine, since the IO penalty for splitting the input file is so severe.
To support this, bcbio-nextgen creates an appropriate type of cluster environment for each step:
IPython parallel provides the distributed framework for creating these processing setups, working on top of existing schedulers like LSF, SGE and TORQUE. It creates processing engines on distributed cores within the cluster, using ZeroMQ to communicate job information between machines.
Cluster schedulers allow this type of control over core usage, but an additional future step is to include memory and disk IO requirements as part of heterogeneous environment creation. Amazon Web Services allows selection of exact memory, disk and compute resources to match the computational step. Eucalyptus and OpenStack bring this control to local hardware and virtual machines.
While the initial alignment and preparation steps require analysis of a full set of reads due to IO and algorithm restrictions, subsequent steps can run with increased parallelism by splitting across genomic regions. Variant detection algorithms do require processing continuous blocks of reads together, allowing local realignment algorithms to correctly characterize closely spaced SNPs and indels. Previously, we’d split analyses by chromosome but this has the downside of tying analysis times to chromosome 1, the largest chromosome.
The pipeline now identifies chromosome blocks without callable reads. These blocks group by either genomic features like repetitive hard to align sequence or analysis requirements like defined target regions. Using the globally shared callable regions across samples, we fraction the genome into more uniform sections for processing. As a result we can work on smaller chunks of reads during time critical parts of the process: applying base recalibration, de-duplication, realignment and variant calling.
A key bottleneck throughout the pipeline is disk usage. Steps requiring reading and writing large BAM or FASTQ files slow down dramatically once they overburden disk IO, distributed filesystem capabilities or ethernet connectivity between storage nodes. A practical solution to this problem is to avoid intermediate files and use unix pipes to stream results between processes.
We reworked our alignment step specifically to eliminate these issues. The previous attempt took a disk centric approach that allowed scaling out to multiple single cores in a cluster. We split an input FASTQ or BAM file into individual chunks of reads, and then aligned each of these chunks independently. Finally, we merged all the individual BAMs together to produce a final BAM file to pass on to the next step in the process. While nicely generalized, it did not scale when running multiple concurrent whole genomes.
The updated pipeline uses multicore support in samtools and aligners like bwa-mem and novoalign to pipe all steps as a stream: preparation of input reads, alignment, conversion to BAM and coordinate sorting of aligned reads. This results in improved scaling at the cost of only being able to increase single sample throughput to the maximum processors on a machine.
More generally, the entire process creates numerous temporary file intermediates that are a cause of scaling issues. Commonly used best-practice toolkits like Picard and GATK primarily require intermediate files. In contrast, tools in the Marth lab’s gkno pipeline handle streaming input and output making it possible to create alignment post-processing pipelines which minimize temporary file creation. As a general rule, supporting streaming algorithms amenable to piping can ameliorate file load issues associated with scaling up variant calling pipelines. This echos the focus on streaming algorithms Titus Brown advocates for dealing with large metagenomic datasets.
While all three of CPU, memory and disk speed limit individual steps during processing, the hardest variable to tweak is disk throughput. CPU and memory limitations have understandable solutions: buy faster CPUs and more memory. Improving disk access is not as easily solved, even with monetary resources, as it’s not clear what combination of disk and distributed filesystem will produce the best results for this type of pipeline.
We’ve experimented with NFS, GlusterFS and Lustre for handling disk access associated with high throughput variant calling. Each requires extensive tweaking and none has been unanimously better for all parts of the process. Much credit is due to John Morrissey and the research computing team at Harvard FAS for help performing incredible GlusterFS and network improvements as we worked through scaling issues, and Glen Otero, Will Cottay and Neil Klosterman at Dell for configuring an environment for NFS and Lustre testing. We can summarize what we’ve learned so far in two points:
Other approaches we’re considering include utilizing high speed local temporary disk, reducing writes to long term distributed storage file systems. This introduces another set of challenges: avoiding stressing or filling up local disk when running multiple processes. We’ve also had good reports about using MooseFS but haven’t yet explored setting up and configuring another distributed file system. I’d love to hear experiences and suggestions from anyone with good or bad experiences using distributed file systems for this type of disk intensive high throughput sequencing analysis.
A final challenge associated with improving disk throughput is designing a pipeline that is not overly engineered to a specific system. We’d like to be able to take advantage of systems with large SSD attached temporary disk or wonderfully configured distributed file systems, while maintaining the ability scale on other systems. This is critical for building a community framework that multiple groups can use and contribute to.
Providing detailed timing estimates for large, heterogeneous pipelines is difficult since they will be highly depending on the architecture and input files. Here we’ll present some concrete numbers that provide more insight into the conclusions presented above. These are more useful as a side by side comparison between approaches, rather than hard numbers to predict scaling on your own systems.
In partnership with Dell Solutions Center, we’ve been performing benchmarking of the pipeline on dedicated cluster hardware. The Dell system has 32 16-core machines connected with high speed InfiniBand to distributed NFS and Lustre file systems. We’re incredibly appreciative of Dell’s generosity in configuring, benchmarking and scaling out this system.
As a benchmark, we use 10x coverage whole genome human sequencing data from the Illumina platinum genomes project. Detailed instructions on setting up and running the analysis are available as part of the bcbio-nextgen example pipeline documentation.
Below are wall-clock timing results, in total hours, for scaling from 1 to 30 samples on both Lustre and NFS fileystems:
| primary | 1 sample | 1 sample | 1 sample | 30 samples | 30 samples | |
|---|---|---|---|---|---|---|
| bottle | 16 cores | 96 cores | 96 cores | 480 cores | 480 cores | |
| neck | Lustre | Lustre | NFS | Lustre | NFS | |
| alignment | cpu/mem | 4.3h | 4.3h | 3.9h | 4.5h | 6.1h |
| align post-process | io | 3.7h | 1.0h | 0.9h | 7.0h | 20.7h |
| variant calling | cpu/mem | 2.9h | 0.5h | 0.5h | 3.0h | 1.8h |
| variant post-process | io | 1.0h | 1.0h | 0.6h | 4.0h | 1.5h |
| total | 11.9h | 6.8h | 5.9h | 18.5h | 30.1h |
Some interesting conclusions:
This is preliminary work as we continue to optimize code parallelism and work on cluster and distributed file system setup. We welcome feedback and thoughts to improve pipeline throughput and scaling recommendations.
Framework for evaluating variant detection methods: comparison of aligners and callers [Blue Collar Bioinformatics] 2013-05-06 08:29
Developing pipelines for detecting variants from high throughput sequencing data is challenging due to rapidly changing algorithms and relatively low concordance between methods. This post will discuss automated methods providing evaluation of variant calls, enabling detailed diagnosis of discordant differences between multiple calling approaches. This allows us to:
This evaluation work is part of a larger community effort to better characterize variant calling methods. A key component of these evaluations is a well characterized set of reference variations for the NA12878 human HapMap genome, provided by NIST’s Genome in a Bottle consortium. The diagnostic component of this work supplements emerging tools like GCAT (Genome Comparison and Analytic Testing), which provides a community platform for comparing and discussing calling approaches.
I’ll show a 12 way comparison between 2 different aligners (novoalign and bwa mem), 2 different post-alignment preparation methods (GATK best practices and the Marth lab’s gkno pipeline), and 3 different variant callers (GATK UnifiedGenotyper, GATK HaplotypeCaller, and FreeBayes). This allows comparison of openly available methods (bwa mem, gkno preparation, and FreeBayes) with those that require licensing (novoalign, GATK’s variant callers). I’ll also describe bcbio-nextgen, the fully automated open-source pipeline used for variant calling and evaluation, which allows others to easily bring this methodology into their own work and extend this analysis.
To compare methods, we called variants on a NA12878 exome dataset from EdgeBio’s clinical pipeline and assessed them against the NIST Genome in a Bottle reference material. Discordant positions where the reference and evaluation variants differ fall into three different categories:
To further identify causes of discordance, we subdivide the missing and extra variants using annotations from the GEMINI variation framework:
We subdivide and restrict our comparisons to help identify sources of differences between methods indistinguishable when looking at total discordant counts. A critical subdivison is comparing SNPs and indels separately. With lower total counts of indels but higher error rates, each variant type needs independent visualization. Secondly, it’s crucial to distinguish between discordance caused by a lack of coverage, and discordance caused by an actual difference in variant assessment. We evaluate only in callable regions with 4 or more reads. This low minimum cutoff provides a valuable evaluation of low coverage regions, which differ the most between alignment and calling methods.
I’ll use this data to provide recommendations for alignment, post-alignment preparation and variant calling. In addition to these high level summaries, the full dataset and summary plots available below providing a starting place for digging further into the data.
We compared two recently released aligners designed to work with longer reads coming from new sequencing technologies: novoalign (3.00.02) and bwa mem (0.7.3a). bwa mem identified 1389 additional concordant SNPs and 145 indels not seen with novoalign. 1024 of these missing variants are in regions where novoalign does not provide sufficient coverage for calling. Of those, 92% (941) have low coverage with less than 10 reads in the bwa alignments. Algorithmic changes impact low coverage regions more due to the decreased evidence and susceptibility to crossing calling coverage thresholds, so we need extra care and consideration of calls in these regions.
Our standard workflow uses novoalign based on its stringency in resolving large insertions and deletions. These results suggest equally good results using bwa mem, along with improved processing times. One caveat to these results is that some of the available Illumina call data that feeds into NIST’s reference genomes comes from a bwa alignment, so some differences may reflect a bias towards bwa alignment heuristics. Using non-simulated reference data sets has the advantage of capturing real biological and process errors, but requires iterative improvement of the reference materials to avoid this type of potential algorithmic bias.
We compared two methods of quality recalibration:
GATK best practice pipelines offer an advantage over the gkno-only pipeline primarily because of improvements in SNP calling from base quality recalibration. Specifically we lose ~1% (824 / 77158) of called variations. These fall into the discordant missing “other” category, so we cannot explain them by metrics such as coverage or genome difficulty. The simplest explanation is that initial poor quality calculations in those regions result in callers missing those variants. Base quality recalibration helps recover them. These results match Brendan O’Fallon’s recent analysis of base quality score recalibration.
This places a practical number on the lost variants when avoiding recalibration either due to scaling or GATK licensing concerns. Some other options for recalibration include Novoalign’s Quality Recalibration and University of Michigan’s BamUtil recab, although we’ve not yet tested either in depth as potential supplements to improve calling in non-GATK pipelines.
For this comparison, we used three general purpose callers that handle SNPs and small indels, all of which have updated versions since our last comparison:
Adjusting variant calling methods has the biggest impact on the final set of calls. Called SNPs differ by 4577 between the three compared approaches, in comparison with aligner and post-alignment preparation changes which resulted in a maximum difference of 1389 calls. This suggests that experimenting with variant calling approaches currently provides the most leverage to improve calls.
A majority of the SNP concordance differences between the three calling methods are in low coverage regions with between 4 and 9 reads. GATK UnifiedGenotyper performs the best in detecting SNPs in these low coverage regions. FreeBayes and GATK HaplotypeCaller both call more conservatively in these regions, generating more potential false negatives. FreeBayes had the fewest heterozygote/homozygote discrimination differences of the three callers.
For indels, FreeBayes and HaplotypeCaller both provide improved sensitivity compared to UnifiedGenotyper, with HaplotypeCaller identifying the most, especially in low coverage regions. In contrast to the SNP calling results, FreeBayes has more calls that match the expected indel but differ in whether a call is a heterozygote or homozygote.
No one caller outperformed the others on all subsets of the data. GATK UnifiedGenotyper performs best on SNPs but is less sensitive in resolving indels. GATK HaplotypeCaller identifies the most indels, but is more conservative than the other callers on SNPs. FreeBayes provides intermediate sensitivity and specificity between the two for both SNPs and indels. A combined UnifiedGenotyper and HaplotypeCaller pipeline for SNPs and indels, respectively, would provide the best overall calling metrics based on this set of comparisons.
Low coverage regions are the key area of difference between callers. Coupled with the alignment results and investigation of variant changes resulting from quality score binning, this suggests we should be more critical in assessing both calls and coverage in these regions. Assessing coverage and potential false negatives is especially critical since we lack good tools to summarize and prioritize genomic regions that are potentially missed during sequencing. This also emphasizes the role of population-based calling to help resolve low coverage regions, since callers can use evidence from multiple samples to better estimate the likelihoods of low coverage calls.
Method comparisons become dated quickly due to the continuous improvement in aligners and variant callers. While these recommendations are useful now, in 6 months there will be new releases with improved approaches. This rapid development cycle creates challenges for biologists hoping to derive meaning from variant results: do you stay locked on software versions whose trade offs you understand, or do you attempt to stay current and handle re-verifying results with every new release?
Our goal is to provide a community developed pipeline and comparison framework that ameliorates this continuous struggle to re-verify. The analysis done here is fully automated as part of the bcbio-nextgen analysis framework. This framework code provides full exposure and revision tracking of all parameters used in analyses. For example, the ngsalign module contains the command lines used for bwa mem and novoalign, as well as all other tools.
To install the pipeline, third-party software and required data files:
wget https://raw.github.com/chapmanb/bcbio-nextgen/master/scripts/bcbio_nextgen_install.py python bcbio_nextgen_install.py /usr/local /usr/local/share/bcbio-nextgen
The installer bootstraps all installation on a bare machine using the CloudBioLinux framework. More details and options are available in the installation documentation.
To re-run this analysis, retrieve the input data files and configuration as described in the bcbio-nextgen example documentation with:
$ mkdir config && cd config $ wget https://raw.github.com/chapmanb/bcbio-nextgen/master/config/\ examples/NA12878-exome-methodcmp.yaml $ cd .. && mkdir input && cd input $ wget https://dm.genomespace.org/datamanager/file/Home/EdgeBio/\ CLIA_Examples/NA12878-NGv3-LAB1360-A/NA12878-NGv3-LAB1360-A_1.fastq.gz $ wget https://dm.genomespace.org/datamanager/file/Home/EdgeBio/\ CLIA_Examples/NA12878-NGv3-LAB1360-A/NA12878-NGv3-LAB1360-A_2.fastq.gz $ wget https://s3.amazonaws.com/bcbio_nextgen/NA12878-nist-v2_13-NGv3-pass.vcf.gz $ wget https://s3.amazonaws.com/bcbio_nextgen/NA12878-nist-v2_13-NGv3-regions.bed.gz $ gunzip NA12878-nist-*.gz $ wget https://s3.amazonaws.com/bcbio_nextgen/NGv3.bed.gz $ gunzip NGv3.bed.gz
Then run the analysis, distributed on 8 local cores, with:
$ mkdir work && cd work $ bcbio_nextgen.py bcbio_system.yaml ../input ../config/NA12878-exome-methodcmp.yaml -n 8
The bcbio-nextgen documentation describes how to parallelize processing over multiple machines using cluster schedulers (LSF, SGE, Torque).
The pipeline and comparison framework are open-source and configurable for multiple aligners, preparation methods and callers. We invite anyone interested in this work to provide feedback and contributions.
We extracted the conclusions for alignment, post-alignment preparation and variant calling from analysis of the full dataset. The visualizations for the full data are not as pretty but we make them available for anyone interested in digging deeper:
The comparison variant calls are also useful for pinpointing algorithmic differences between methods. Some useful subsets of variants:
We encourage reanalysis and welcome suggestions for improving the presentation and conclusions in this post.
Bioinformatics open source interoperability Hackathon at the Broad Institute [Blue Collar Bioinformatics] 2013-04-08 15:25
On April 7th and 8th, a group of biologists and programmers gathered at the Broad Institute to work on improving interoperability of open-source bioinformatics tools. Organized by the Open Bioinformatics Foundation and GenomeSpace team, this was part of the lead up to the Bioinformatics Open Source Conference (BOSC) in July in Berlin. The event is part of an ongoing series of coding sessions (Codefests or Hackathons) organized by the open bioinformatics community, which give programmers who typically work together remotely a chance to code and discuss in the same place for two days. These have been successful in both producing new code and in building connections which help sustain development of these community projects.
One major challenge in analyzing biological data is interfacing multiple bioinformatics tools. Tools often work independently, and where general architectures like plugins or API exist they are often project specific. This results in isolated islands of data exchange, but transferring data or resources between tools requires work that is often rate-limiting or insurmountable.
Our goal at the hackathon was to provide simple APIs and implementations that help facilitate transfers between multiple islands of functionality. GenomeSpace does this by providing a central hub and API to push and pull from tools. We wanted to generalize this to support multiple tools, and build client implementations that demonstrate this in practice. The long term goal is to encourage tool developers to provide server side APIs compatible with the more general library, making extension of the connector toolkit easier. For developers, the client API would allow them to easily transfer files between multiple tools without needing to learn and implement the specific transfer APIs of each tool.
We called this high level client library Genome Connector (gcon, for short) and took a practical approach by implementing client libraries that provide a common interface to multiple tools: GenomeSpace, Galaxy, BaseSpace, 23andMe and general key-value stores through jClouds. To identify a reasonable amount of work for two days, we focused on file transfer: authentication, finding files, getting and putting files to remote analysis platforms. In addition we defined some critical components for doing biological work:
We also identified other useful extensions that would help improve interoperability and facilitate building connected tools, like providing Publish/subscribe hooks to avoid having to poll servers for updates, and smarter approaches to sending data to avoid duplication and unnecessary transfer of data.
The output of our discussion and coding are common Genome Connector implementations in multiple languages. GitHub repositories are available for in-progress Java, Python and Clojure implementations. These wrap multiple diverse tools and expose them through a common top level API, allowing developers to push and pull data from multiple tools.
I’m immensely grateful to the incredible participants who generously donated their time and expertise to help with these projects. For anyone interested we also have detailed documentation on discussions during the hackathon.
If you’re a bioinformatics programmers interested in open source coding and helping answer biological questions by improving usability and connectivity of tools, you’re welcome to join the OpenBio and BOSC communities. We’ve created a biological interoperability mailing list for additional discussion. The next BOSC conference is July 19th and 20th in Berlin, Germany as part of the ISMB conference. There will also be another two day Codefest proceeding BOSC on July 17th and 18th. Abstracts for talks at BOSC are due this Friday, April 12th. Looking forward to seeing everyone at future BOSC and coding events.
The influence of reduced resolution quality scores on alignment and variant calling [Blue Collar Bioinformatics] 2013-02-13 05:49
We have a large upcoming whole genome sequencing project with Illumina, and they approached us about delivering BAM files with reduced resolution base quality scores. They have a white paper describing the approach, which involves binning scores to reduce resolution. This reduces the number of scores describing the quality of a base from 40 down to 8.
The advantage of this approach is a significant reduction in file size. BAM files use BGZF compression, and the underlying gzip DEFLATE algorithm compresses based on shared text regions. Reducing the number of quality values increases shared blocks and improves compression. This reduces BAM file sizes by 25-35%: an exome BAM file reduced from 5.7Gb to 3.7Gb after quality binning.
The potential downside is that the reduction in quality resolution may impact alignment and variant calling approaches that rely on base quality scores. To assess this, I implemented quality score binning as part of the bcbio-nextgen analysis pipeline using the CRAM toolkit and ran alignment, recalibration, realignment and variant calling on:
A comparison of alignment and variant calls from the three approaches indicates that binning has nearly no impact on alignment and a small impact on variant calls, primarily in low depth regions.
We aligned 100bp paired end reads with Novoalign, a quality aware aligner. Comparison of mapped reads showed nearly no impact on total mapped reads. The plot below shows a generic delta of changes in mapped reads across the 22 autosomes alongside the increase in unmapped pairs. Out of 73 million total reads, the changes account for ~0.003% of the total reads. There also did not appear to be any worrisome patterns of loss for specific chromosomes. Overall, there is a minimal impact of quality score binning on the ability to align the reads.
We called variants using the GATK Unified Genotyper following the best practice recommendations for exomes and then compared calls from original and binned quality scores. Both approaches for binning — pre-binning, and pre-binning plus post-quality recalibration binning — showed similar levels of concordance to non-binned quality scores: 99.81 and 99.78, respectively. Since the additional binning after recalibration provides a smaller prepared BAM file for storage and has a similar impact to pre-binning only, we used this for additional analysis of discordant variants.
The table below shows the discordant differences between the 40 quality score resolution and binned, 8 quality score resolution BAMs. 40 quality discordant variants are those called with full quality score resolution but not called, or called differently, after binning to 8 quality score resolution. Conversely, the 8-quality discordants are those called uniquely after quality binning:
| Overall genotype concordance | 99.78 |
| concordant: total | 117887 |
| concordant: SNPs | 109144 |
| concordant: indels | 8743 |
| 40-quality discordant: total | 821 |
| 40-quality discordant: SNPs | 759 |
| 40-quality discordant: indels | 62 |
| 8-quality discordant: total | 1289 |
| 8-quality discordant: SNPs | 1240 |
| 8-quality discordant: indels | 49 |
| het/hom discordant | 259 |
We investigated the discordant variants further since 1.5% of the total variant calls change as a result of binning, Of the 1851 unique discordant variants, approximately half (928) fall into reproducible variants identified by looking at ensemble combinations of replicates. Of these potentially problematic discordant variants more than half are in low coverage regions with less than 10 reads:
The major influence of quality score binning is resolution of variants in low coverage regions. This manifests as differences in heterozygote and homozygote calling, indel representation and filtering differences related to quality and mappability. To assess the potential impact, we looked at the loss in callable bases on a 30x whole genome sequence when moving from a minimum of 5 reads to a minimum of 10, using GATK’s CallableLoci tool. Regions with read coverage of 5 to 9 make up 4.7 million genome positions, 0.17% of the total callable bases.
| 5 read minimum | 10 read minimum | |
|---|---|---|
| Callable bases | 2,775,871,235 | 2,771,109,000 |
| Percent callable | 96.90% | 96.73% |
| Low coverage | 17,641,980 | 22,404,215 |
| No coverage/ poor mapping | 71,272,008 | 71,272,008 |
In conclusion, quality score binning provides a useful reduction in input file sizes with minimal impact on alignment. For variant calling, use additional caution in low coverage regions with less than 10 supporting reads. Given the rapid increases in read throughput that are driving the need for file size reduction, quality score binning is a worthwhile tradeoff for high-coverage recalling work.
An automated ensemble method for combining and evaluating genomic variants from multiple callers [Blue Collar Bioinformatics] 2013-02-06 07:25
A key goal of the Archon Genomics X Prize infrastructure is development of a set of highly accurate reference genome variants. I’ve described our work preparing these reference genomes, and specifically defined the challenges behind merging genomic variant calls from multiple technologies and calling methods. Comparing calls from two different calling methods, for example GATK and samtools mpileup, produces a large number of differing variants which need reconciliation. Taking the overlapping subset from multiple callers is too conservative and will miss real variations, while including all calls is too liberal and introduces false positives.
Here I’ll describe a fully automated approach for preparing an accurate set of combined variant calls. Ensemble machine learning methods are a powerful way to incorporate inputs from multiple models. We use a heuristic and support vector machine (SVM) algorithm to consolidate variants, producing a final set of calls with better sensitivity and specificity than current best practice methods. The approach is open source, fully automated and generalizable to both human diploid sequencing as well as X Prize haploid reference fosmids.
We use a pair of replicates from EdgeBio’s clinical exome sequencing pipeline to prepare ensemble variant calls in the widely studied HapMap NA12878 genome. Compared to variants from a single calling method, the ensemble method produced more concordant variants when comparing the replicates, with fewer discordants. The finalized ensemble calls also provide a useful method to compare strengths and weaknesses of individual calling methods. The implementation is freely available and I’ll discuss how to get it running on your data so you can use, critique and extend the methods. This work is a collaboration between Harvard School of Public Health, EdgeBio and NIST.
A difficult aspect of evaluating variant calling methods is establishing a reference set of calls. For X Prize we use three established methods, each of which comes with tradeoffs. Metrics like transition/transversion ratios or dbSNP overlap provide a global picture of calling but are not fine grained enough to distinguish improvements over best practices. Sanger validation restricts you to a manageable subset of calls. Comparisons against public resources like 1000 genomes bias results towards technologies and callers used in preparing those variant callsets.
Here we employ a fourth method by comparing replicates from EdgeBio’s clinical exome sequencing pipeline. These are NA12878 samples independently prepared using Nimblegen’s version 3.0 kit and sequenced on an Illumina HiSeq. By comparing the replicates in regions with 4 or more reads in both samples, we identify the ability of variant calling algorithms to call identical variations with differing coverage and error profiles.
We aligned reads with novoalign and performed deduplication, base recalibration and realignment using GATK best practices. With these prepared reads, we called variants with five approaches:
We took a combined heuristic and machine learning approach to consolidate these five sets of variant calls into a final ensemble callset. The first step is to prepare the union of all variant calls from the input callers, identifying calling methods that support each variant. Secondly, we annotate each variant with metrics including strand bias, allele balance, regional sequence entropy, position of calls within reads, regional base quality and overall genotype likelihoods. We then filter this prepared set of all possible variants to produce a final set of trusted calls.
The first filtering step is to heuristically identify trusted variants based on the number of callers supporting them. This configurable parameter allow you to make an initial conservative cutoff for including variants in the final calls: I trust variants supported by N or more callers.
For the remaining calls that fall below the trusted support cutoff, we distinguish true and false positives using a support vector machine (SVM). The annotated metrics described above are the input parameters and we prepare true and false positives for the classifier using a multi-step process:
The final set of calls includes the trusted variants and those that pass the SVM filtering. An input configuration file for variant preparation and assessment allows adjustment of the trusted threshold as well as defining which metrics to use for building the SVM classifiers.
We assess calling sensitivity and specificity by comparing concordant and discordant variant calls between the replicates. To provide a consistent method to measure both SNP and indel correctness, we use the positive predictive value as the percentage of concordant calls between duplicates (concordant variants / (concordant variants + discordant variants)). This is different than the overall concordance rate, which also includes non-variant regions where both replicates do not call a variation. As a result these percentages will be lower if you expect the 99% values that result when including reference calls. The advantage of this metric is that it’s easily interpreted as the percentage of concordant called variants. It also allows individual comparisons of SNPs and indels, since the overall number of indels are low compared to the total bases considered. GATK’s VariantEval documentation has a nice discussion of alternative metrics to genotype concordance.
As a baseline we used calls from GATK’s UnifiedGenotyper to represent a current best practice approach. GATK calls 117079 SNPs, 86.6% of which are concordant. It also calls 14966 indels, with 64.6% concordant. Here are the full concordant and discordant numbers, broken down by variant type and replicate:
| concordant: total | 111159 |
| concordant: SNPs | 101495 |
| concordant: indels | 9664 |
| rep1 discordant: total | 9857 |
| rep1 discordant: SNPs | 7514 |
| rep1 discordant: indels | 2343 |
| rep2 discordant: total | 11029 |
| rep2 discordant: SNPs | 8070 |
| rep2 discordant: indels | 2959 |
| het/hom discordant | 4181 |
Our ensemble method produces improvements in both total concordant variants detected and the ratio of concordant to discordants. For SNPs, the ensemble calls add 5345 additional variants to a total of 122424, with an increase in concordance to 87.4%. For indels the major improvement is in removal of discordants: We identify 14184 indels with 67.2% concordant. Here is the equivalent table for the ensemble method:
| concordant: total | 116608 |
| concordant: SNPs | 107063 |
| concordant: indels | 9545 |
| rep1 discordant: total | 9555 |
| rep1 discordant: SNPs | 7581 |
| rep1 discordant: indels | 1974 |
| rep2 discordant: total | 10445 |
| rep2 discordant: SNPs | 7780 |
| rep2 discordant: indels | 2665 |
| het/hom discordant | 3975 |
For scientists who have worked to increase sensitivity and specificity of individual variant callers, it’s exciting to be able to improve both simultaneously. As mentioned above, you can also tune the method to increase specificity or sensitivity by adjusting the support needed for including trusted variants.
The final ensemble callsets from both replicates are available as VCF files from GenomeSpace in the xprize/NA12878-exome-v_03 folder:
Calling the same samples with multiple callers allows direct comparisons between calling methods. The advantage of producing an accurate final set of ensemble calls is that this provides a baseline to evaluate the strengths and weaknesses of different calling methods. The figure below compares concordant, missing variants and additional variants called by each of the 5 methods in comparison with the consolidated ensemble calls:
Variant calling methods with recommendations for both calling and filtering provide the best out of the box performance. An advantage of GATK and samtools is they provide calling, variant quality metrics, and filtering. On the other side, FreeBayes is a good example of a powerful tool that takes some time to learn to filter optimally. One potential source of bias in producing the individual calls is that I personally have more experience with GATK tools so may have room to improve with the other callers.
Combining multiple calling approaches improves both sensitivity and specificity of the final set of variants. The downside is the need to run and coordinate calls from all of the different callers. To mitigate this, we developed an automated pipeline that ties together multiple open-source tools using two custom components:
bcbio-nextgen has a script, built on functionality in the CloudBioLinux project, that automates installation of associated variant callers and data dependencies:
wget https://raw.github.com/chapmanb/bcbio-nextgen/master/scripts/bcbio_nextgen_install.py python bcbio_nextgen_install.py install_directory data_directory
With the dependencies installed, you describe the input files and analysis with a YAML formatted input file. The NA12878 ensemble configuration file used for this analysis provides a useful starting point. Run the analysis, distributed on multiple cores, with:
bcbio_nextgen.py bcbio_system.yaml ensemble_sample.yaml -n 8
The bcbio-nextgen documentation provides additional details about configuration inputs and distributed processing. The framework generally handles the automation and processing involved with high throughput sequencing analysis.
EdgeBio kindly made the NA12878 datasets used in this analysis publicly available:
I welcome feedback on the approach, data or tools and am actively working to extend this and make it easier to use. As re-sequencing becomes increasingly important for human health applications it is critical that we develop open, shared best-practice workflows to handle the data processing. This allows us to focus back on the fun and difficult work of understanding the biology.
Genomics X Prize public phase update: variant classification and de novo calling [Blue Collar Bioinformatics] 2012-09-17 08:41
Last month I described our work at HSPH and EdgeBio preparing reference genomes for the Archon Genomics X Prize public phase, detailing methods used in the first version of our NA19239 variant calls. We’ve been steadily improving the calling approaches, and released version 0.2 on the X Prize validation website and GenomeSpace. Here I’ll describe the improvements we’ve made over the last month, focusing on two specific areas:
Our goal is to iteratively improve our calling and variant preparation to create the best possible set of reference calls. I’d be happy to talk more with anyone working on similar problems or with insight into useful ways to improve our current callsets. We have a Get Satisfaction site for discussion and feedback, and have offered a $2500 prize for helpful comments.
As a reminder, all of the code and data used here is freely available:
Public/chapmanb/xprize/NA19239-v0_2. Public/EdgeBio/PublicData/Release1. de novo variant calling performs reference-free assembly of either local or global genome regions, then subsequently uses these assemblies to call variants relative to a known reference. The advantage is that assemblies can avoid errors associated with mapping to the reference, helping resolve large variations as well as small variations near problem alignments or low complexity regions.
Hybrid approaches that use localized de novo assembly in variant regions help mitigate the extensive computational requirements associated with whole-genome assembly. Complete Genomics variant calling and GATK 2.0′s Haplotype Caller both provide pipelines for hybrid de novo assembly in variant detection. The fermi and SGA assemblers are also used in variant calling, although the paths from assembly to variants are not as automated.
Thanks to Zam’s generous assistance, we used cortex_var for localized de novo assembly and variant calling within individual fosmid boundaries. As a result, CloudBioLinux now contains automated build instructions for cortex_var , handling binary builds for multiple k-mer and color combinations. An automated cortex_var pipeline, part of the bcbio-nextgen toolkit, runs the processing workflow:
Directly comparing GATK and cortex_var calls shows similar levels of concordance and discordance as the GATK/samtools comparison from the last post:
| concordant: total | 153787 |
| concordant: SNPs | 130913 |
| concordant: indels | 22874 |
| GATK discordant: total | 20495 |
| GATK discordant: SNPs | 6522 |
| GATK discordant: indels | 13973 |
| cortex_var discordant: total | 26790 |
| cortex_var discordant: SNPs | 21342 |
| cortex_var discordant: indels | 5448 |
11% of the GATK calls and 14% of the cortex_var calls are discordant. The one area where cortex_var does especially well is on indels: 19% of the cortex_var indels do not agree with GATK, in comparison with 37% of the GATK calls and 25% of the samtools calls. The current downside to this is SNP calling, where cortex_var has 3 times more discordant calls than GATK.
Overlapping variant calls from different calling methods (GATK, FreeBayes, samtools and cortex_var) and sequencing technologies (Illumina, SOLiD and IonTorrent) produces 170,286 potential calls in the fosmid regions. 58% (99,227) of these are present in all callers and technologies, so we need to do better than the intersection in creating a consolidated callset.
As detailed in the previous post, we filter the full set in two ways. The first is to keep trusted variants based on their presence in a defined number of technologies or callers. We currently have an inclusive set of criteria: keep variants present in either 4 out of the 7 callsets, 2 distinct technologies, or 3 distinct callers. This creates a trusted set containing 95% (162,202) of the variants. Longer term the goal is to reduce the trusted count and rely on automated filtering approaches based on input metrics.
This second automated filtering step uses a support vector machine (SVM) to evaluate the remaining variants. We train the SVM on potential true positives from variants that overlap in all callers and technologies, and potential false positives found uniquely in one single caller. The challenge is to find useful metrics associated with these training variants that will help provide discrimination.
In version 0.1 we filtered with a vanilla set of metrics: depth and variant quality score. To identify additional metrics, we used a great variant visualization tool developed by Keming Labs alongside HSPH and EdgeBio. I’ll write up more details about the tool once we have a demonstration website but the code is already available on GitHub.
To remove variants preferentially associated with poorly mapping or misaligned reads, we identified two useful metrics. ReadPosEndDist, written as a GATK annotation by Justin Zook at NIST, identifies variants primarily supported by calls at the ends of reads. Based on visual examination, these associate with difficult to map regions, as identified by Genome Mappability Scores:
Secondly, we identified problematic allele balances that differ from the expected ratios. For haploid fosmid calls, we expect 100% of reads to support variants and 0% to support reference calls (in diploid calls, you also need to handle heterozygotes with 50% expected allele balance). In practice, the distribution of reads can differ due to sequencer and alignment errors. We use a metric that measures deviation from the expected allele balance and associates closely with variant likelihoods:
To assess the influence of adding de novo calls and additional filtering metrics on the resulting call set, we compare against whole genome Illumina and Complete Genomics calls for NA19239. Previously we’d noticed two major issues during this comparison: a high percentage of discordant indel calls and a technology bias signaled by better concordance with Illumina than Complete.
The comparison between fosmid and Illumina data shows a substantial improvement in the indel bias. Previously 46% of the total indel calls were discordant, indicative of a potential false positive problem. With de novo calls and improved filtering, we’ve lowered this to only 10% of the total calls.
| concordant: total | 147684 |
| concordant: SNPs | 133861 |
| concordant: indels | 13823 |
| fosmid discordant: total | 7519 |
| fosmid discordant: SNPs | 5856 |
| fosmid discordant: indels | 1663 |
| Illumina discordant: total | 5640 |
| Illumina discordant: SNPs | 1843 |
| Illumina discordant: indels | 3797 |
This improvement comes with a decrease in the total number of concordant indel calls, since we moved from 17,816 calls to 13,823. However a large number of these seemed to be Illumina specific since 60% of the previous calls were discordant when compared with Complete Genomics. The new callset reduces this discrepancy and only 22% of the indels are now discordant:
| concordant: total | 139155 |
| concordant: SNPs | 127243 |
| concordant: indels | 11912 |
| fosmid discordant: total | 15484 |
| fosmid discordant: SNPs | 12028 |
| fosmid discordant: indels | 3456 |
| Complete Genomics discordant: total | 7311 |
| Complete Genomics discordant: SNPs | 4972 |
| Complete Genomics discordant: indels | 2273 |
These comparisons provide some nice confirmation that we’re moving in the right direction on filtering. As before, we extract potential false positives and false negatives to continue to refine the calls: potential false positives are those called in the fosmid dataset and in neither of the Illumina or Complete Genomics sets. Potential false negatives are calls that both Illumina and Complete agree on that the fosmid calls lack.
In the new callsets, there are 5,499 (3.5%) potential false positives and 1,422 (0.9%) potential false negatives. We’ve reduced potential false positives in the previous set from 10% with a slight increase in false negatives. These subsets are available along with the full callset on GenomeSpace. We’re also working hard on an NA12878 callset with equivalent approaches and will make that available soon for community feedback.
I hope this discussion, open source code, and dataset release is useful to everyone working on problems of improving variant calling accuracy and filtering. I welcome feedback on calling, consolidation methods, interesting metrics to explore, machine learning or any of the other topics discussed here.
Genomics X Prize public phase: reference genome preparation and comparisons to Illumina and Complete Genomics [Blue Collar Bioinformatics] 2012-08-15 09:10
The Archon Genomics X Prize, presented by Express Scripts, is a 10 million dollar competition to establish highly accurate clinical grade sequencing and variation detection methods. Our group at Harvard School of Public Health works with the EdgeBio team on developing the infrastructure for the competition: identify variations in the grading genomes and provide software to compare these reference variation sets against a competitor’s list of variations.
The exciting aspect of the Genomics X Prize is that it enables open comparisons between sequencing technologies and variant calling methodologies. Sequencing genomes to the high degree of accuracy sufficient for clinical usage is a difficult, open, problem. Here I’ll present detailed numbers comparing variants called by different sequencing technologies and variant callers.
The public phase of the Genomics X Prize starts today, August 15th. The goal of this six month period is to have an open dialog with everyone working in the sequencing and variant calling communities. We want to refine our methods to provide the most accurate and fair variant calling for the reference genomes. To start the discussion we’ve prepared:
The goal of this writeup, and the X Prize public phase, is to iterate over calling and unification methods to improve our algorithms and approaches. Rather than promoting or disparaging any particular technology or calling method, we’re instead providing full transparency and a good-faith effort to combining approaches. Our hope is that this will help engage the community, encourage feedback, and result in a unbiased and accurate set of reference genomes for the competition.
For the August 15th public phase kickoff, we prepared a reference data set of NA19239 based on pooled sequencing of haploid fosmid clones. The callable regions of these clones totaled 129,513,026 total bases, covering ~4% of the 3.1 billion bases in the human genome. We use fosmid clones to obtain complete regional haplotype coverage and focus on partial genome coverage to achieve high coverage depth and accuracy for assessed regions.
Version 0.1 of the NA19239 reference set uses variant calls from two technologies: Illumina and SOLiD; and three callers: GATK’s Unified Genotyper, FreeBayes and SAMtools. To move from these data to a unified call set we:
The challenging decisions begin when merging and filtering the final call set. This requires careful bookkeeping and variant representation to ensure identical variants are directly comparable, followed by setting cutoffs for variant inclusion.
The details of variant comparisons introduce an additional layer of complexity during assessment. The approach we’ve taken is create a normalized set of variants so all comparison differences are due to actual call differences rather than variant representation. We split multiple nucleotide polymorphisms into individual calls, split complex indel-variant combinations, and left-align remaining variants.
For haploid/diploid comparisons, we establish haplotype blocks for the diploid sequence based on phasing provided in the input variant file, and then compare the best matching haplotype to our fosmid reference. Single nucleotide polymorphisms and indels less than 30bp require exact machines between two comparison genomes. Larger indels and structural variations receive more flexible matching with confidence intervals around start and end coordinates.
The goal of the normalized, compared variants is to reflect real underlying differences in calling approaches relative to how well we can currently resolve variation endpoints.
For a concrete example of two different variant calling approaches, below is a table comparing GATK variants against samtools calls for the NA19239 sample, using identically aligned and post-processed BAMs:
| concordant: total | 160851 |
| concordant: SNPs | 136146 |
| concordant: indels | 24705 |
| GATK discordant: total | 13925 |
| GATK discordant: SNPs | 1315 |
| GATK discordant: indels | 12610 |
| samtools discordant: total | 25368 |
| samtools discordant: SNPs | 17247 |
| samtools discordant: indels | 8121 |
The number of discordant variant calls is high, making up 8% of the GATK calls and 14% of the samtools calls, and samtools calls almost 16,000 additional SNPs compared to GATK. As a result, a large percentage of variants require making hard decisions: are those additional calls interesting, real variants in samtools and false negatives in the GATK calls? Or conversely, are they false positives in samtools that GATK correctly excludes?
There is a similar level of discrepancy when comparing variant calls between Illumina and SOLiD sequencing. Below is a comparison between GATK Unified genotyper calls on the two technologies:
| concordant: total | 135263 |
| concordant: SNPs | 122267 |
| concordant: indels | 12996 |
| Illumina discordant: total | 39491 |
| Illumina discordant: unique | 7079 |
| Illumina discordant: SNPs | 15188 |
| Illumina discordant: indels | 24303 |
| SOLiD discordant: total | 16022 |
| SOLiD discordant: unique | 3800 |
| SOLiD discordant: SNPs | 3908 |
| SOLiD discordant: indels | 12114 |
Unique coverage explains some differences: 4% of the Illumina variants (7079) and 2.5% (3800) of the SOLiD variants were uniquely covered by the technologies. However, the remaining variant discordant calls are on the order of those seen in the technology comparisons. Adding to the complexity, we find only 84% of the total concordant variants compared to the Illumina only GATK/samtools comparison.
The level of discrepancy between calling methods and sequencing approaches introduces complexity in the preparation of the final call set: How much evidence does a variant need for inclusion? Can single calls be true positives if supported by high confidence values? This will require extensive refinement throughout the public phase. For the initial version 0.1 release of NA19239, we took the following high level approach to filtering:
The result is a unified call set of 171,009 variants derived from all technologies and callers, that we’re releasing as NA19239 version 0.1.
To assess the quality of the unified call set, we compared to two public genomes:
This provides us with three independent call sets to assess variability between approaches. To provide a baseline, here is the comparison of the Illumina and Complete Genomics calls in our assessment regions:
| Overall genotype concordance | 98.47 |
| concordant: total | 205868 |
| concordant: SNPs | 186365 |
| concordant: indels | 19503 |
| Illumina discordant: total | 31267 |
| Illumina discordant: SNPs | 19334 |
| Illumina discordant: indels | 11933 |
| Complete Genomics discordant: total | 15174 |
| Complete Genomics discordant: SNPs | 9586 |
| Complete Genomics discordant: indels | 5510 |
We see familiar discordance rates: 13% of the Illumina calls and 7% of the Complete Genomics calls differ. Since it’s diploid versus diploid, this comparison includes all heterozygous variant matches. As a result the numbers in this comparison will be higher, but it is a good order of magnitude approximation for looking at our fosmid reference set versus each individual technology.
The comparison against the Illumina whole genome variant calls contains 12% discordant calls in our fosmid reference set, with 79% of those being indel differences. Indels are notoriously more difficult to identify and assess, so this will be an area of increased focus as we move forward:
| concordant: total | 150420 |
| concordant: SNPs | 132604 |
| concordant: indels | 17816 |
| fosmid discordant: total | 19624 |
| fosmid discordant: SNPs | 4165 |
| fosmid discordant: indels | 15459 |
| Illumina discordant: total | 5475 |
| Illumina discordant: SNPs | 2952 |
| Illumina discordant: indels | 2523 |
The Complete Genomics comparison has 17% discordant calls including 2x more discordant SNP calls. This highlights another key area of call set refinement: identifying and correcting for technology specific calls.
| concordant: total | 139559 |
| concordant: SNPs | 126296 |
| concordant: indels | 13263 |
| fosmid discordant: total | 29883 |
| fosmid discordant: SNPs | 10162 |
| fosmid discordant: indels | 19721 |
| Complete Genomics discordant: total | 7571 |
| Complete Genomics discordant: SNPs | 5542 |
| Complete Genomics discordant: indels | 1965 |
The initial NA19239 public genome for the Genomics X Prize provides unified variant calls based on two sequencing technologies and three calling methods. I’ve delved into a lot of details on our approaches, challenges and goals with the hopes of encouraging suggestions from other researchers working on these problems. We’re especially interested in feedback on these areas of ongoing research:
The call sets used here are available as public data folders on GenomeSpace:
Combined with the open source code and configurations, we hope this will provided interested researchers with all the raw materials needed to reproduce and extend these analyses. Your feedback and suggestions are very welcome.
Extending the GATK for custom variant comparisons using Clojure [Blue Collar Bioinformatics] 2012-03-04 20:13
The Genome Analysis Toolkit (GATK) is a full-featured library for dealing with next-generation sequencing data. The open-source Java code base, written by the Genome Sequencing and Analysis Group at the Broad Institute, exposes a Map/Reduce framework allowing developers to code custom tools taking advantage of support for: BAM Alignment files through Picard, BED and other interval file formats through Tribble, and variant data in VCF format.
Here I’ll show how to utilize the GATK API from Clojure, a functional, dynamic programming language that targets the Java Virtual Machine. We’ll:
The Clojure variation library is freely available and is part of a larger project to provide variant assessment capabilities for the Archon Genomics XPRIZE competition.
GATK’s well documented Map/Reduce API eases the development of custom programs for processing BAM and VCF files. The presentation from Eli Lilly is a great introduction to developing your own custom GATK Walkers in Java. Here we’ll follow a similar approach to code these in Clojure.
We’ll start by defining a simple Java base class that extends the base GATK walker and defines an output and input variable. The output is a string specifying the output file to write to, and the input is any type of variant file the GATK accepts. Here we’ll be dealing with VCF input files:
public abstract class BaseVariantWalker extends RodWalker {
@Output
public String out;
@ArgumentCollection
public StandardVariantContextInputArgumentCollection invrns = new StandardVariantContextInputArgumentCollection();
}
This base class is all the Java we need. We implement the remaining walker in Clojure and will walk through the fully annotated source in sections. To start, we import the base walker we wrote and extend this to generate a Java class, which the GATK will pick up and make available as a command line walker:
(ns bcbio.variation.vcfwalker (:import [bcbio.variation BaseVariantWalker]) (:gen-class :name bcbio.variation.vcfwalker.VcfSimpleStatsWalker :extends bcbio.variation.BaseVariantWalker))
Since this is a Map/Reduce framework, we first need to implement the map function. GATK passes this function a tracker, used to retrieve the actual variant call values and a context which describes the current location. We use the invrns argument we defined in Java to reference the input VCF file supplied on the commandline. Finally, we extract the quality score from each VariantContext and return those. This map function produces a stream of quality scores from the input VCF file:
(defn -map
[this tracker ref context]
(if-not (nil? tracker)
(for [vc (map from-vc
(.getValues tracker (.variants (.invrns this))
(.getLocation context)))]
(-> vc :genotypes first :qual))))
For the reduce part, we take the stream of quality scores and plot a histogram. In the GATK this happens in 3 functions: reduceInit starts the reduction step and creates a list to add the quality scores to, reduce collects all of the quality scores into this list, and onTraversalDone plots a histogram of these scores using the Incanter statistical library:
(defn -reduceInit
[this]
[])
(defn -reduce
[this cur coll]
(if-not (nil? cur)
(vec (flatten [coll cur]))
coll))
(defn -onTraversalDone
[this result]
(doto (icharts/histogram result
:x-label "Variant quality"
:nbins 50)
(icore/save (.out this) :width 500 :height 400)))
We’ve implemented a full GATK walker in Clojure, taking advantage of existing Clojure plotting libraries. To run this, compile the code into a jarfile and run like a standard GATK tool:
$ lein uberjar
$ java -jar bcbio.variation-0.0.1-SNAPSHOT-standalone.jar -T VcfSimpleStats
-r test/data/grch37.fa --variant test/data/gatk-calls.vcf --out test.png
which produces a plot of quality score distributions:
GATK’s Variant Annotator is a useful way to add metrics information to a file of variants. These metrics allow filtering and prioritization of variants, either by variant quality score recalibration or hard filtering. We can add new annotation metrics by inheriting from GATK Java interfaces. Here we’ll implement Mean Neighboring Base Quality (NBQ), a metric from the Atlas2 variation suite that assesses the quality scores in a region surrounding a variation.
We start walking through the full implementation by again defining a generated Java class that inherits from a GATK interface. In this case, InfoFieldAnnotation:
(ns bcbio.variation.annotate.nbq
(:import [org.broadinstitute.sting.gatk.walkers.annotator.interfaces.InfoFieldAnnotation]
[org.broadinstitute.sting.utils.codecs.vcf VCFInfoHeaderLine VCFHeaderLineType])
(:require [incanter.stats :as istats])
(:gen-class
:name bcbio.variation.annotate.nbq.MeanNeighboringBaseQuality
:extends org.broadinstitute.sting.gatk.walkers.annotator.interfaces.InfoFieldAnnotation))
The annotate function does the work of calculating the mean quality score. We define functions that use the GATK API to:
With these three functions, we can use the Clojure threading macro to cleanly organize the steps of the operation as we retrieve the pileup, get the qualities and calculate the mean:
(defn -annotate
[_ _ _ _ contexts _]
(letfn [(get-pileup [context]
(if (.hasExtendedEventPileup context)
(.getExtendedEventPileup context)
(.getBasePileup context)))
(neighbor-qualities [[offset read]]
(let [quals (-> read .getBaseQualities vec)]
(map #(nth quals % nil) (range (- offset flank-bp) (+ offset flank-bp)))))
(pileup-qualities [pileup]
(map neighbor-qualities (map vector (.getOffsets pileup) (.getReads pileup))))]
{"NBQ" (->> contexts
vals
(map get-pileup)
(map pileup-qualities)
flatten
(remove nil?)
istats/mean
(format "%.2f"))}))
With this in place we can now run this directly using the standard GATK command line arguments. As before, we create a jar file with the new annotator, and then pass the name as a desired annotation when running the VariantAnnotator, producing a VCF file with NBQ annotations:
$ lein uberjar
$ java -jar bcbio.variation-0.0.1-SNAPSHOT-standalone.jar -T VariantAnnotator
-A MeanNeighboringBaseQuality -R test/data/GRCh37.fa -I test/data/aligned-reads.bam
--variant test/data/gatk-calls.vcf -o annotated-file.vcf
In addition to extending the GATK through walkers and annotations you can also utilize the extensive API directly, taking advantage of parsers and data structures to handle common file formats. Using Clojure’s Java interoperability, the variantcontext module provides a high level API to parse and extract information from VCF files. To loop through a VCF file and print the location, reference allele and called alleles for each variant we:
with-open statement to ensure closing of the resource.VariantContext maps for each variant in the file.
(use 'bcbio.variation.variantcontext)
(with-open [vcf-source (get-vcf-source "test/data/gatk-calls.vcf")]
(doseq [vc (parse-vcf vcf-source)]
(println (:chr vc) (:start vc) (:ref-allele vc)
(-> vc :genotypes first :alleles)))
This produces:
MT 73 #<Allele G*> [#<Allele A> #<Allele A>]
MT 150 #<Allele T*> [#<Allele C> #<Allele C>]
MT 152 #<Allele T*> [#<Allele C> #<Allele C>]
MT 195 #<Allele C*> [#<Allele T> #<Allele T>]
I hope this tour provides some insight into the powerful tools that can be rapidly built by leveraging the GATK from Clojure. The full library contains a range of additional functionality including normalization of complex MNPs and support for phased haplotype comparisons.
Slides and replay of my “Using R with Hadoop” webinar now available #rstats #hadoop [Things I tend to forget] 2013-01-25 17:22
I owe a big “thank you” to all of you who attended my webinar yesterday “Using R with Hadoop”. Revolution Analytics partnered with us at Think Big Analytics to produce the webinar, and I owe them thanks as well.
For those of you who missed it, the slides and replay are now available from Revolution Analytics.
How to specify a MapR distro when launching Elastic MapReduce clusters with the Ruby CLI [Things I tend to forget] 2013-01-09 13:17
Amazon’s Elastic MapReduce Ruby client allows you to specify which of the supported Hadoop distributions to use, currently either Amazon’s Apache 1.0.3-based distribution or MapR’s M3 and M5 editions.
I found the CLI’s option documented at <http://docs.aws.amazon.com/ElasticMapReduce/latest/DeveloperGuide/emr-mapr.html>:
To launch an Amazon EMR job flow with MapR using the CLI
Set the –with-supported-products parameter to either mapr-m3 or mapr-m5 to run your job flow on the corresponding version of the MapR Hadoop distribution.
The following example launches a job flow running with the M3 Edition of MapR.
elastic-mapreduce –create –alive \
–instance-type m1.xlarge –num-instances 5 \
–with-supported-products mapr-m3
For additional information about launching job flows using the CLI, see the instructions for each job flow type in Create a Job Flow.
Slides from “Tapping the Data Deluge with R” lightning talk #rstats #PAWCon [Things I tend to forget] 2012-10-02 11:56
Here is my presentation from last night’s Boston Predictive Analytics Meetup graciously hosted by Predictive Analytics World Boston.
The talk is meant to provide an overview of (some) of the different ways to get data into R, especially supplementary data sets to assist with your analysis.
All code and data files are available at github: http://bit.ly/pawdata (https://github.com/jeffreybreen/talk-201210-data-deluge)
The slides themselves are on slideshare: http://bit.ly/pawdatadeck (http://www.slideshare.net/jeffreybreen/tapping-the-data-deluge-with-r)
Slides from today’s Big Data Step-by-Step Tutorials: Infrastructure series and Intro to R+Hadoop with RHadoop’s rmr [Things I tend to forget] 2012-03-10 12:13
Here are my presentations from today’s Boston Predictive Analytics Big Data Workshop.
All code and config files are available at github: https://github.com/jeffreybreen/tutorial-201203-big-data
My portion of the workshop was divided into four parts, three focusing on different infrastructure scenarios and ending with a deep dive into the rmr R package:
Not everyone has Big Data. Some of us have an occasional need to analyze a data set larger than comfortably fits in our existing analysis environment either due to disk, CPU, or memory constraints. For these times, launching a single, large machine in the cloud may fit the bill. This part of presentation walks through how to launch just such a machine using Amazon’s EC2 cloud computing platform. Since I tend to run R and RStudio on Linux, that’s the focus of this tutorial, but the general outline may be helpful to others as well.
Scale up using the cloud. The Apache Whirr cloud management tool makes it easy to launch a Hadoop cluster on EC2. We use the Cloudera VM from presentation #1 as a launching point for the cluster and, thanks to a Whirr-generated proxy script, submit jobs and fetch results from our local VM just as before. For extra credit, we see how Whirr can save us money by bidding for excess capacity via EC2′s spot instances.
Crunching Big Data with R. Originally a Java-only ecosystem, Hadoop Streaming allows the creation of mappers, reducers, and combiners in any language which can handle stdin and stdout—but that doesn’t mean you want to have to write code to manage I/O at that level. After a quick (and undoubtedly incomplete) survey of Hadoop-related R packages, we walk through some of the abstractions and features of RHadoop’s rmr package which make it easier for R developers to get started. We walk through a sample mapper and reducer, demonstrating and documenting the native R objects which carry the data from step to step.
Thank you to the session’s sponsors, all the speakers, and to an interesting and engaged audience. Special thanks to John Versotek for arranging such an informative and enjoyable day, and for the opportunity to take part.
Use geom_rect() to add recession bars to your time series plots #rstats #ggplot [Things I tend to forget] 2011-08-15 23:50
Zach Mayer’s work reproducing John Hussman’s Recession Warning Composite prompted me to dig this trick out of my (Evernote) notebook.
First, let’s grab some data to plot using the very handy getSymbols() function from Jeffrey Ryan’s quantmod package. We’ll load the U.S. unemployment rate (UNRATE) from the St. Loius Fed’s Federal Reserve Economic Data (src="FRED") and load the time series into a data.frame:
unrate = getSymbols('UNRATE',src='FRED', auto.assign=F)
unrate.df = data.frame(date=time(unrate), coredata(unrate) )
Now FRED provides a USREC time series which we could use to draw the recessions. It’s a bit awkward, though, as it contains a boolean to flag recession months since January 1921. All we really want are the start and end dates of each recession. Fortunately, the St. Louis Fed publishes just such a table on their web site. (See the answer to “What dates are used for the US recession bars in FRED graphs?” on http://research.stlouisfed.org/fred2/help-faq/.) Sometimes it’s still easier to cut-and-paste (and the static table covers another 64 years, go figure):
recessions.df = read.table(textConnection(
"Peak, Trough
1857-06-01, 1858-12-01
1860-10-01, 1861-06-01
1865-04-01, 1867-12-01
1869-06-01, 1870-12-01
1873-10-01, 1879-03-01
1882-03-01, 1885-05-01
1887-03-01, 1888-04-01
1890-07-01, 1891-05-01
1893-01-01, 1894-06-01
1895-12-01, 1897-06-01
1899-06-01, 1900-12-01
1902-09-01, 1904-08-01
1907-05-01, 1908-06-01
1910-01-01, 1912-01-01
1913-01-01, 1914-12-01
1918-08-01, 1919-03-01
1920-01-01, 1921-07-01
1923-05-01, 1924-07-01
1926-10-01, 1927-11-01
1929-08-01, 1933-03-01
1937-05-01, 1938-06-01
1945-02-01, 1945-10-01
1948-11-01, 1949-10-01
1953-07-01, 1954-05-01
1957-08-01, 1958-04-01
1960-04-01, 1961-02-01
1969-12-01, 1970-11-01
1973-11-01, 1975-03-01
1980-01-01, 1980-07-01
1981-07-01, 1982-11-01
1990-07-01, 1991-03-01
2001-03-01, 2001-11-01
2007-12-01, 2009-06-01"), sep=',',
colClasses=c('Date', 'Date'), header=TRUE)
Now the only “gotcha” is that our recession data start long before our unemployment data, so let’s trim it to match:
recessions.trim = subset(recessions.df, Peak >= min(unrate.df$date) )
Finally, we use ggplot2′s geom_line() layer to draw the unemployment data and transparent (alpha=0.2) pink rectangles to overlay the recessions:
g = ggplot(unrate.df) + geom_line(aes(x=date, y=UNRATE)) + theme_bw() g = g + geom_rect(data=recessions.trim, aes(xmin=Peak, xmax=Trough, ymin=-Inf, ymax=+Inf), fill='pink', alpha=0.2)
One-liners which make me love R: twitteR’s searchTwitter() #rstats [Things I tend to forget] 2011-07-21 11:00
R reminds me a lot of English. It’s easy to get started, but very difficult to master. So for all those times I’ve spent… well, forever… trying to figure out the “R way” of doing something, I’m glad to share these quick wins.
My recent R tutorial on mining Twitter for consumer sentiment wouldn’t have been possible without Jeff Gentry’s amazing twitteR package (available on CRAN). It does so much of the behind-the-scenes heavy lifting to access Twitter’s REST APIs, that one line of code is all you need to perform a search and retrieve the (even paginated) results:
library(twitteR)
tweets = searchTwitter("#rstats", n=1500)
You can search for anything, of course, “#rstats” is just an example. (And if you’re really into that hashtag, the twitteR package even provides an Rtweets() function which hardcodes that search string for you.) The n=1500 specifies the maximum number of tweets supported by the Search API, though you may retrieve fewer as Twitter’s search indices contain only a couple of days’ tweets.
What you get back is a list of tweets (technically “status updates”):
> head(tweets) [[1]] [1] "Cloudnumberscom: CloudNumbers.com \023 #Rstats gets real in the cloud http://t.co/Vw4Gupr via @AddToAny" [[2]] [1] "0_h_r_1: CloudNumbers.com \023 #Rstats gets real in the cloud via DecisionStats - I came across Cloudnumbers.com . ... http://tinyurl.com/5sjagjg" [[3]] [1] "cmprsk: RT I just joined the beta to run #Rstats in the cloud with cloudnumbers.com http://t.co/lvVp0YJ via @cloudnumberscom http://bit.ly/lbSruR" [[4]] [1] "0_h_r_1: I just joined the beta to run #Rstats in the cloud with cloudnumbers.com http://t.co/lvVp0YJ via @cloudnumberscom" [[5]] [1] "cmprsk: RT man, the #rstats think people I am too soft on #sas, the #sas people think I am too soft on #wps, the #wps pe... http://bit.ly/innEv8" [[6]] [1] "keepstherainoff: Thanks to @cmprsk @geoffjentry and @MikeKSmith for colour-coded #Rstats GUI advice" > class(tweets[[1]]) [1] "status" attr(,"package") [1] "twitteR"
Now that you have some tweets, the fun really begins. To get you started, the status class includes a very handy toDataFrame() accessor method (see ?status):
> library(plyr) > tweets.df = ldply(tweets, function(t) t$toDataFrame() )

> str(tweets.df) 'data.frame': 131 obs. of 10 variables: $ text : Factor w/ 122 levels "CloudNumbers.com \023 #Rstats gets real in the cloud http://t.co/Vw4Gupr via @AddToAny",..: 1 2 3 4 5 6 7 8 9 10 ... $ favorited : logi NA NA NA NA NA NA ... $ replyToSN : logi NA NA NA NA NA NA ... $ created : POSIXct, format: "2011-07-04 13:50:39" "2011-07-04 13:48:10" "2011-07-04 13:29:00" "2011-07-04 13:23:42" ... $ truncated : logi FALSE FALSE FALSE FALSE FALSE FALSE ... $ replyToSID : logi NA NA NA NA NA NA ... $ id : Factor w/ 131 levels "87941406873751552",..: 1 2 3 4 5 6 7 8 9 10 ... $ replyToUID : logi NA NA NA NA NA NA ... $ statusSource: Factor w/ 17 levels "<a href="http://twitter.com/tweetbutton" rel="nofollow">Tweet Button</a>",..: 1 2 3 1 3 4 5 5 3 4 ... $ screenName : Factor w/ 64 levels "Cloudnumberscom",..: 1 2 3 2 3 4 2 5 3 6 ...
You can pull a particular user’s tweets just as easily with the userTimeline() function. Heck, the package even lets you tweet from R if you use Jeff’s companion ROAuth package, but that requires more than one line….
Enjoy!
Use Dropbox’s public folder for web publishing via Notepad (or emacs or…) [Things I tend to forget] 2011-07-19 12:12
Remember The Good Old Days when all you needed to host a web site was a file system and Notepad (or emacs or TeachText)?
Well, I do, and I can’t say that I miss them… until last week when I tried to insert the JavaScript for some motion charts into a WordPress.com post. It’s impossible. Literally. Don’t waste your time. Seriously.
Self-hosted WordPress blogs can use some custom field hackery, but there’s no such option for us easy-way-out WordPress.com users.
Just save your HTML page to your “Public” directory in Dropbox and it will get its own public URL which you can find in Dropbox’s context menu:

It’s not the ideal embedding I was hoping for — WordPress.com even strips out iframes — but it’s quick and easy and does the job.
One-liners which make me love R: Make your data dance (Hans Rosling style) with googleVis #rstats [Things I tend to forget] 2011-07-15 00:42
It may be a cliché, but much of R’s utility comes from its amazing community. And by community, I am specifically referring to the bright, hard-working people who are willing to share their knowledge and code with the rest of us. Because of their contributions, we can do some amazingly cool and useful things with very little code of our own. It is in this context that I launch this new series to highlight packages and functions which make it easy to do jaw-droppingly cool and useful things.
First up: the googleVis package by Markus Gesmann and Diego de Castillo which makes it easy — often with just one-line of R — to harness the Google Visualization API. Annotated timelines, gauges, maps, org charts, tree maps, and more are suddenly at your command.
I’m going to focus on the motion chart, popularized by Hans Rosling in his groundbreaking 2006 TED talk on global economic development. (If you haven’t seen it yet, you should. Right now. Seriously. Go.) Motion charts are an innovative way to display multidimensional time series in an interactive way. And the googleVis package even comes with some sample data to make it even easier to try them out.
The package is available from CRAN if you need to install it.
To get started, load the package and the included “Fruits” data.frame:
library(googleVis) data(Fruits)
This data.frame contains some sample data about sales of various fruits at different locations for different years. There’s even a proper Date column already constructed for us from the numeric Year column:

To make the chart, we need to give the gvisMotionChart() function our data.frame and tell it a few things about it: the column which identifies the items to examine (idvar=Fruit), the time dimension (timevar=Date), and optionally a name to use to identify the chart in the generated HTML and JavaScript (we’ll use chartid="ILoveFruits"):
M = gvisMotionChart(data=Fruits, idvar="Fruit", timevar="Date", chartid="ILoveFruit")
That’s it.
You can view your chart with the overridden plot() function. It will automatically spawn a browser window and serve up your chart through R’s internal web server:
plot(M)
Since WordPress doesn’t allow embedded JavaScript, please click through to see the motion chart in action:

You can also access all 165 lines of the generated HTML and JavaScript and save it to disk:
cat(unlist(M$html), file="output/ILoveFruits.html")
Time suck alert: googleVis may make them easy to create, but motion charts can be a lot of fun to play with. You have been warned…
If you want to take a look at an example with some real data, you might be interested in the 20 Years of the U.S. Domestic Airline Market In 20 seconds post on my work blog.
Finally, here are the slides from my lightning talk on this topic at this month’s Greater Boston useR Group meeting:
Have fun!
installing R 2.13.1 on Amazon EC2′s “Amazon Linux” AMI #rstats [Things I tend to forget] 2011-07-08 14:27
Condensed from this post (and comments) on David Chudzicki’s blog, tweaked, and updated for R-2.13.1.
Assumes you’re starting with a virgin “Amazon Linux” AMI. I picked “Basic 64-bit Amazon Linux AMI 2011.02.1 Beta” (AMI Id: ami-8e1fece7) because it was marked as free tier eligible on the “Quick Start” tab of AWS’s “Launch Instance” dialog box:
$ sudo yum -y install make libX11-devel.* libICE-devel.* libSM-devel.* libdmx-devel.* libx* xorg-x11* libFS* libX* readline-devel gcc-gfortran gcc-c++ texinfo tetex $ wget http://cran.r-project.org/src/base/R-2/R-2.13.1.tar.gz $ tar zxf R-2.13.1.tar.gz && cd R-2.13.1 $ ./configure && make $ # make coffee... or finish your PhD thesis... (yes, it takes that long) [...] $ # finally, if all is well: $ sudo make install $ cd $ R --version R version 2.13.1 (2011-07-08) Copyright (C) 2011 The R Foundation for Statistical Computing ISBN 3-900051-07-0 Platform: x86_64-unknown-linux-gnu (64-bit) R is free software and comes with ABSOLUTELY NO WARRANTY. You are welcome to redistribute it under the terms of the GNU General Public License version 2. For more information about these matters see http://www.gnu.org/licenses/.
As always, refer to the Installation and Administration manual for details and options.
If you want to install RCurl, or anything which depends on it like twitteR, you’ll need to install libcurl & friends first:
$ sudo yum -y install libcurl libcurl-devel
slides from my R tutorial on Twitter text mining #rstats [Things I tend to forget] 2011-07-04 14:56
Update: An expanded version of this tutorial will appear in the new Elsevier book Practical Text Mining and Statistical Analysis for Non-structured Text Data Applications by Gary Miner et. al which is now available for pre-order from Amazon.
In conjunction with the book, I have cleaned up the tutorial code and published it on github.
Last month I presented this introduction to R at the Boston Predictive Analytics MeetUp on Twitter Sentiment.
The goal of the presentation was to expose a first-time (but technically savvy) audience to working in R. The scenario we work through is to estimate the sentiment expressed in tweets about major U.S. airlines. Even with a tiny sample and a very crude algorithm (simply counting the number of positive vs. negative words), we find a believable result. We conclude by comparing our result with scores we scrape from the American Consumer Satisfaction Index web site.
Jeff Gentry’s twitteR package makes it easy to fetch the tweets. Also featured are the plyr, ggplot2, doBy, and XML packages. A real analysis would, no doubt, lean heavily on the tm text mining package for stemming, etc.
Here is the slimmed-down version of the slides:
And here’s a PDF version to download.
Special thanks to John Verostek for putting together such an interesting event, and for providing valuable feedback and help with these slides.
Update: thanks to eagle-eyed Carl Howe for noticing a slightly out-of-date version of the score.sentiment() function in the deck. Missing was handling for NA values from match(). The deck has been updated and the code is reproduced here for convenience:
score.sentiment = function(sentences, pos.words, neg.words, .progress='none')
{
require(plyr)
require(stringr)
# we got a vector of sentences. plyr will handle a list
# or a vector as an "l" for us
# we want a simple array ("a") of scores back, so we use
# "l" + "a" + "ply" = "laply":
scores = laply(sentences, function(sentence, pos.words, neg.words) {
# clean up sentences with R's regex-driven global substitute, gsub():
sentence = gsub('[[:punct:]]', '', sentence)
sentence = gsub('[[:cntrl:]]', '', sentence)
sentence = gsub('\\d+', '', sentence)
# and convert to lower case:
sentence = tolower(sentence)
# split into words. str_split is in the stringr package
word.list = str_split(sentence, '\\s+')
# sometimes a list() is one level of hierarchy too much
words = unlist(word.list)
# compare our words to the dictionaries of positive & negative terms
pos.matches = match(words, pos.words)
neg.matches = match(words, neg.words)
# match() returns the position of the matched term or NA
# we just want a TRUE/FALSE:
pos.matches = !is.na(pos.matches)
neg.matches = !is.na(neg.matches)
# and conveniently enough, TRUE/FALSE will be treated as 1/0 by sum():
score = sum(pos.matches) - sum(neg.matches)
return(score)
}, pos.words, neg.words, .progress=.progress )
scores.df = data.frame(score=scores, text=sentences)
return(scores.df)
}
We’re only 10% human. According to…who? [What You're Doing Is Rather Desperate] 2013-10-30 18:49
Reading an interesting post at Genomes Unzipped, “Human genetics is microbial genomics“, which states:
Only 10% of cells on your “human” body are human anyway, the rest are microbial.
Have you read a sentence like that before? So have I. So has a reader who left a comment:
I was wondering if you have a source for “Only 10% of cells on your “human” body are human anyway, the rest are microbial”
It’s a good question. Everyone quotes this figure, almost no-one provides a reference. Let’s go in search of one.
Wikipedia. Don’t make it your primary source. However, it’s often a good place to start. From the human microbiome article:
Bacterial cells are much smaller than human cells, and there are at least ten times as many bacteria as human cells in the body (approximately 1014 versus 1013).[7] [8]
Reference [8]: Berg, RD (1996). The indigenous gastrointestinal microflora. Trends Microbiol. 4 (11): 430–435. If you have access to the full text, you’ll find a PDF which seems to be a poor-quality scan of the printed article. From page 432:
In summary, there are ten viable indigenous bacteria in the GI tract for every cell in the human body: 1013 total GI bacteria compared with 1012 total cells making up the human body.
Note the order of magnitude differences compared with the Wikipedia article. I was unable to find a reference in this article to any paper where these numbers were measured or estimated. And so…
…reference [7]: Savage, DC (1977). Microbial Ecology of the Gastrointestinal Tract. Ann. Rev. Microbiol. 31: 107-133.
Another PDF so old that we cannot copy/paste from it. The opening sentences:
The adult human organism is said to be composed of approximately 1013 eukaryotic animal cells (27). That statement is only an expression of a particular point of view. The various body surfaces and the gastrointestinal canals of humans may be colonized by as many
as 1014 indigenous prokaryotic and eukaryotic microbial cells (70).
Note the order of magnitude discrepancies compared with the previous article.
Reference (27) is: Dobzhansky, T. (1971). Genetics of the Evolutionary Process, Vol. 1. New York: Columbia Univ. We’ll leave that one there.
Reference (70) is: Luckey, TD (1972). Am. J. Clin. Nutr. 25: 1292-95. Time to search PubMed:
luckey td[au] 1972[dp]
It’s the first entry, titled “Introduction to intestinal microecology” and it’s freely-available.
Hey look, another ancient PDF – and the last page is 1294, not 1295. First page, second paragraph:
The composition of this system is surprising. Adult man carries 1012 microbes associated with his epidermis and 1014 microbes in his alimentary tract (Fig. 1). The latter number is based upon 1011 microbes/g contents of an alimentary tract with a capacity of approximately 1 liter. The 1013 cells (2) in his body are a distinct numerical minority of the total being that we call man. If we abandon anthropomorphism for the microbic view, we must admire the efficiency of these microbes in using man as a vehicle to further their own cause.
This would seem to be the “definitive” reference, for now. Reference (2) in that quote is Dobzhansky, again.
No mention of adult woman. It’s OK, that’s just how people spoke and wrote in the 1970s.
Bacteria and Alzheimer’s disease: I just need to know if ten patients are enough [What You're Doing Is Rather Desperate] 2013-10-29 23:41
You can guarantee that when scientists publish a study titled:
a newspaper will publish a story titled:
Without access to the paper, it’s difficult to assess the evidence. I suggest you read Jonathan Eisen’s analysis of the abstract. Essentially, it makes two claims:
Regardless of the biochemistry – which does not sound especially convincing to me[1] – how about the statistics?
LPS was detected in 0/10 normal brains, compared with 4/10 AD brains. The “tl;dr” version of this discussion – if you think that those look like rather small numbers, you’re correct.
We can set up a matrix in R to contain those values.
ad <- matrix(c(4, 6, 0, 10), nrow = 2)
colnames(ad) <- c("AD", "Norm")
rownames(ad) <- c("lps+", "lps-")
ad
# AD Norm
# lps+ 4 0
# lps- 6 10
Are those proportions significantly different? Or to put it another way: “I just need to know if 3 (10) patients are enough.” Let’s talk about statistical power.
Without going too deeply into the mathematics, the power of a statistical test is a number between 0 and 1, which describes the probability of a type II (false negative) error. For example, when power = 0.8, the probability of a false negative (concluding no difference between groups when in fact, there is one) is 0.2.
We’re looking at proportions in two groups (a two-proportion test), where the power depends on several parameters:
R provides us with the function power.prop.test():
Usage:
power.prop.test(n = NULL, p1 = NULL, p2 = NULL, sig.level = 0.05,
power = NULL,
alternative = c("two.sided", "one.sided"),
strict = FALSE)
How it works: you set one of the parameters n, p1, p2, sig.level or power to NULL and it is calculated from the other parameters.
To get started – what’s the power of the study in the publication, using sig.level = 0.05?
ppt <- power.prop.test(n = 10, p1 = 0, p2 = 0.4) ppt$power # [1] 0.6250675
Effectively, what that means is that the probability of a false negative (concluding no difference in LPS detection between normal and AD brains when there is a difference) is about 0.375. That’s rather high.
Most researchers set power = 0.8 as an acceptable threshold. So – how many samples per group do we need to achieve power = 0.8 at sig.level = 0.05?
ppt <- power.prop.test(p1 = 0, p2 = 0.4, power = 0.8) ppt$n # [1] 14.45958
About 15 samples. Not many more than 10 – but more than 10, nevertheless. How about something more stringent: power = 0.9, sig.level = 0.01?
ppt <- power.prop.test(p1 = 0, p2 = 0.4, power = 0.9, sig.level = 0.01) ppt$n # [1] 27.16856
Note that with larger sample sizes (e.g. 100 per group), the proportion of normal brains containing LPS can be quite high compared with AD brains and still be significant (at p = 0.05):
ppt <- power.prop.test(p2 = 0.4, power = 0.8, n = 100) ppt$p1 # [1] 0.2180086
Finally, a plot showing the increase of power with group sample size at p = 0.05, p = 0.01 (click for larger version):
library(ggplot2)
# quick, dirty, ugly, but it works
df1 <- data.frame(n = 10:30,
p = sapply(10:30, function(x) power.prop.test(p1 = 0, p2 = 0.4, n = x)$power),
s = 0.05)
df2 <- data.frame(n = 10:30,
p = sapply(10:30, function(x) power.prop.test(p1 = 0, p2 = 0.4, n = x, sig.level = 0.05)$power),
s = 0.05)
df3 <- rbind(df1, df2)
ggplot(df3) + geom_point(aes(n, p, color = factor(s))) + theme_bw()
So much for power. How about testing for a difference between the groups?
You might be tempted to reach for the two-proportion test, implemented in R as prop.test(). You should not – but here’s the result anyway:
prop.test(ad) 2-sample test for equality of proportions with continuity correction data: c(0, 4) out of c(10, 10) X-squared = 2.8125, df = 1, p-value = 0.09353 alternative hypothesis: two.sided 95 percent confidence interval: -0.803636315 0.003636315 sample estimates: prop 1 prop 2 0.0 0.4 Warning message: In prop.test(c(0, 4), c(10, 10)) : Chi-squared approximation may be incorrect
Note the warning message. That’s telling you that we don’t have enough samples for the calculation to be informative. The so-called Cochran conditions stipulate that no cell should contain a count of zero and more than 80% of cells should have counts of at least 5. Some power calculators, such as this one, will tell you when these assumptions have been violated.
The alternative is Fisher’s exact test:
fisher.test(ad)
Fisher's Exact Test for Count Data
data: ad
p-value = 0.08669
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
0.7703894 Inf
sample estimates:
odds ratio
Inf
In summary then: not only is the conclusion that “LPS from periodontal bacteria can access the AD brain during life” rather premature, but this study “lacks power”. We cannot say whether LPS detection in the AD brains is significantly different to that in normal brains.
[1] and I used to be a biochemist (D. Phil., 1997)
Web scraping using Mechanize: PMID to PMCID/NIHMSID [What You're Doing Is Rather Desperate] 2013-09-16 22:55
Web services are great. Pass them a URL. Structured data comes back. Parse it, analyse it, visualise it. Done.
Web scraping – interacting programmatically with a web page – is not so great. It requires more code and when the web page changes, the code breaks. However, in the absence of a web service, scraping is better than nothing. It can even be rather satisfying. Early in my bioinformatics career the realisation that code, rather than humans, can automate the process of submitting forms and reading the results was quite a revelation.
In this post: how to interact with a web page at the NCBI using the Mechanize library.
I would like to know if I can convert PMID to NIHMSID, if there is no PMCID associated with that PMID and retrieve both of them if PMCID exists. I want to do this programmatically may be using eutils if possible. I know about this link http://www.ncbi.nlm.nih.gov/pmc/pmctopmid/ that will do the work but its not programmatically. I also tried the services by PMC http://www.ncbi.nlm.nih.gov/pmc/tools/oai/#examples but not much of success.
Some definitions. PMID = PubMed ID, an identifier for articles in the PubMed database. PMCID, a similar identifier for articles deposited in PubMed Central. NIHMSID – articles in PubMed Central have one of these identifiers if submitted via the NIH manuscript submission system.
The NCBI provide this web page, but no web service, for converting PMID to PMCID/NIHMSID or PMCID to PMID.
Enter Mechanize. I know of libraries for Perl, Python and Ruby – there may be others. Here’s how to use the Ruby version to submit a form and retrieve the results.
First, load the library and fetch the web page:
require 'mechanize'
agent = Mechanize.new
page = agent.get("http://www.ncbi.nlm.nih.gov/pmc/pmctopmid/")
If you view the page on the Web, you’ll see that it contains 2 forms: one for a general search at NCBI, the other for ID conversion. You can verify this by searching the Mechanize::Page object for forms and pretty-printing the second one:
puts page.search("form").count
# 2
pp page.search("form")[1]
# output not shown
Next, we want to set values in the form. Let’s get the second form and examine the fields:
form = page.forms[1] pp form
#<Mechanize::Form
{name nil}
{method "POST"}
{action "/pmc/pmctopmid/"}
{fields
[hidden:0xfa4150 type: hidden name: p$a value: ]
[hidden:0xfa3fe8 type: hidden name: p$l value: PAFAppLayout]
[hidden:0xfa3e80 type: hidden name: p$st value: pmc]
[hidden:0xfa3d2c type: hidden name: SessionId value: F4FC1F49237BDA11_0186SID]
[hidden:0xfa3bc4 type: hidden name: Snapshot value: /projects/PMC/PMCStatic@2.60]
[textarea:0xd543dc type: name: PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.Ids value: ]}
{radiobuttons
[radiobutton:0xfa4a10 type: radio name: PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.from_db value: from_pmid]
[radiobutton:0xfa4880 type: radio name: PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.from_db value: from_pmcid]}
{checkboxes
[checkbox:0xfa459c type: checkbox name: PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.ToFile value: false]}
{file_uploads}
{buttons
[submit:0xfa4704 type: submit name: Clipboard value: Get IDs from PubMed clipboard]
[submit:0xfa4420 type: submit name: ConvertButton value: Convert]
[submit:0xfa42cc type: submit name: ClearButton value: Clear]}>
Compare that with the web page or its source code. We want to do the following:
There are various ways to do all of those things:
# select the radio button by value form.radiobutton_with(:value => "from_pmid").check # select the text area by name and set values form["PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.Ids"] = "21707345\n23482678" # select the CSV checkbox by name form.checkbox_with(:name => "PAFAppLayout.AppController.Page.PMCToPmidC.MainPortlet.ToFile").check
Here PMIDs are separated by newline but you could also use commas, spaces, semicolons or vertical bars.
Finally, submit the form. There are 3 buttons in the form; we want the second one which is named ConvertButton and has value Convert:
f = agent.submit(form, form.buttons[1]) f.save "ids.txt"
Result – a file named ids.txt, with these contents:
PMID,PMCID,NIHMSID 21707345,-,NIHMSID331689 23482678,PMC3592971,NIHMSID392341
My tip: study the web page and its source code carefully, making note of the names and values for the elements of interest. Those elements will then be much easier to find and alter when you come to work with the Mechanize object representation.
Microarrays, scan dates and Bioconductor: it shouldn’t be this difficult [What You're Doing Is Rather Desperate] 2013-08-22 01:16
When dealing with data from high-throughput experimental platforms such as microarrays, it’s important to account for potential batch effects. A simple example: if you process all your normal tissue samples this week and your cancerous tissue samples next week, you’re in big trouble. Differences between cancer and normal are now confounded with processing time and you may as well start over with new microarrays.
Processing date is often a good surrogate for batch and it was once easy to extract dates from Affymetrix CEL files using Bioconductor. It seems that this is no longer the case.
Once upon a time (about 10 months ago), I could write code to process CEL files from the Affymetrix Human Exon 1.0 ST array that looked like this:
library(simpleaffy) # assuming a covdesc file in same directory as CEL files exp.cel <- read.affy(path = "data/celfiles") exp.cel@cdfName <- "exon.pmcdf" exp.rma <- rma(exp.cel)
Getting the scan date could not have been easier:
pd <- pData(protocolData(exp.rma)) head(pd) # ScanDate # HCT_CON_A.CEL 2009-12-11T02:57:33Z # HCT_CON_B.CEL 2009-12-11T00:56:07Z # HCT_Treated_A.CEL 2009-12-11T04:27:12Z # HCT_Treated_B.CEL 2009-12-11T01:26:20Z # HT29_CON_A.CEL 2009-12-11T03:27:22Z # HT29_CON_B.CEL 2009-12-11T03:56:58Z
Alas, this code now fails at the first hurdle (reading CEL files):
Error in read.affybatch(filenames = l$filenames, phenoData = l$phenoData, : The affy package is not designed for this array type. Please use either the oligo or xps package. FALSE
Fair enough. I’ve used the oligo package before, so we try that instead:
library(oligo)
# assuming CEL files in ./data/celfiles
exp.cel <- read.celfiles(list.celfiles("data/celfiles", full.names = T))
exp.rma <- rma(exp.cel)
Wait – what happened to the dates?
pd <- pData(protocolData(exp.rma)) head(pd) # exprs dates # HCT_CON_A.CEL data/celfiles//HCT_CON_A.CEL # HCT_CON_B.CEL data/celfiles//HCT_CON_B.CEL # HCT_Treated_A.CEL data/celfiles//HCT_Treated_A.CEL # HCT_Treated_B.CEL data/celfiles//HCT_Treated_B.CEL # HT29_CON_A.CEL data/celfiles//HT29_CON_A.CEL # HT29_CON_B.CEL data/celfiles//HT29_CON_B.CEL # oligo has a runDate() method # no joy there either runDate(exp.cel) # [1] # Levels:
OK – let’s look at xps. A prerequisite for installation is something called ROOT; not the super user, but a data analysis framework. If you’re on an Ubuntu-like system, ignore all the confusing advice around the Web about setting environment variables and just:
sudo apt-get install root-system
After xps installation we read the manual:
In contrast to other packages, which rely on the Bioconductor method for creating cdf environments, we need to create ROOT scheme files directly from the Affymetrix source files, which need to be downloaded first from the Affymetrix web site. However, once created, it is in principle possible to distribute the ROOT scheme files, too.
I’m sorry. There are only so many hoops that I’m willing to jump through in order to simply read from a file and this is a step too far.
So my current fix – use apt-cel-convert, part of the Affymetrix Power Tools suite, to convert to text and then grep for dates in the format MM/DD/YY:
apt-cel-convert -f text -o . myfile.CEL grep ^DatHeader myfile.CEL | grep -ioP "\b\d+\/\d+\/\d+\b" # 01/15/13
I don’t know yet whether those regular expressions work for all cases.
Have a better suggestion? Let’s hear it.
Update – for completeness I’m using R 3.0.1, Bioconductor 2.12, oligo 1.24.2 on Ubuntu 10.04. I’ve also used a CEL file from a different platform, HG U133A, which gave this:
runDate(exp.cel) # [1] [0..46114] 10434-10425 #799 133A 8-222-02:CLS=4733 RWS=4733 XIN=3 YIN=3 VE=17 # 2.0 08/22/02 12:08:07 \024 \024 HG-U133A.1sq \024 \024 \024 \024 \024 \024 \024 # \024 \024 6 # Levels: [0..46114] 10434-10425 #799 133A 8-222-02:CLS=4733 RWS=4733 XIN=3 YIN=3 VE=17 # 2.0 08/22/02 12:08:07 \024 \024 HG-U133A.1sq \024 \024 \024 \024 \024 \024 \024 # \024 \024 6
i.e. the entire DatHeader line has been stored as a factor.
Interestingly: the sentence adverbs of PubMed Central [What You're Doing Is Rather Desperate] 2013-07-15 18:20
Scientific writing – by which I mean journal articles – is a strange business, full of arcane rules and conventions with origins that no-one remembers but to which everyone adheres.
I’ve always been amused by one particular convention: the sentence adverb. Used with a comma to make a point at the start of a sentence, as in these examples:
Surprisingly, we find that the execution of karyokinesis and cytokinesis is timely…
Grossly, the tumor is well circumscribed with fibrous capsule…
Correspondingly, the short-term Smad7 gene expression is graded…
The example that always makes me smile is interestingly. “This is interesting. You may not have realised that. So I said interestingly, just to make it clear.”
With that in mind, let’s go looking for sentence adverbs in article abstracts.
1. Download PubMed Central
Code and data for this post can be found at Github.
We need abstracts. One source of these is the PubMed Central (PMC) archive at the NCBI FTP site. Create a directory on your system to hold the files (e.g. db/pmc), move to it and:
wget http://ftp.ncbi.nlm.nih.gov/pub/pmc/articles.A-B.tar.gz
wget http://ftp.ncbi.nlm.nih.gov/pub/pmc/articles.C-H.tar.gz
wget http://ftp.ncbi.nlm.nih.gov/pub/pmc/articles.I-N.tar.gz
wget http://ftp.ncbi.nlm.nih.gov/pub/pmc/articles.O-Z.tar.gz
find ./ -name "*.tar.gz" -exec tar zxvf {} \;
Caution: the compressed archives are 1.5 – 2.5 GB and will take some time to download. They uncompress to about 47 GB of storage (at the time of writing).
2. Extract the sentence adverbs
The PMC files are in XML format. I’m not especially expert in XML parsing but when required, I reach for Ruby’s nokogiri. Here’s the complete code:
#!/usr/bin/ruby
require "nokogiri"
f = File.open(ARGV[0])
doc = Nokogiri::XML(f)
f.close
ameta = doc.xpath("//article/front/article-meta")
pmc = ameta.xpath("//article-id[@pub-id-type='pmc']").text.chomp
epub = ameta.xpath("//pub-date[@pub-type='epub']/year").text.chomp
ppub = ameta.xpath("//pub-date[@pub-type='ppub']/year").text.chomp
abs = ameta.xpath("//abstract").text.chomp
titl = ameta.xpath("//title-group/article-title").text.chomp
jour = doc.xpath("//article/front/journal-meta/journal-id[@journal-id-type='nlm-ta']").text.chomp
abs.scan(/[A-Z][a-z]+ly,/m) {
b = $~.begin(0)
e = $~.end(0)
a = $&.gsub(",", "")
o = [pmc, jour.downcase, epub, ppub, a, b, e]
puts o.join(",")
}
The code uses XPath expressions to pull out abstracts and some other metadata from the PMC NXML files. It then searches the abstract for words that end with “ly,”. For reasons to be explained later, journal abbreviations are converted to lower-case and the position of the match is also recorded.
Save it as pmcly.rb and run it on the directory of PMC files like so:
find db/pmc -name "*.nxml" -exec pmcly.rb {} > adverbs.csv \;
This takes some time – overnight on my home machine. The result (first 10 lines of 92 940):
2751461,aaps j,2008,NA,Alternatively,350,364 2751463,aaps j,2008,NA,Unfortunately,315,329 2844510,aaps j,2010,NA,Generally,125,135 2751449,aaps j,2008,NA,Ideally,581,589 2751387,aaps j,2008,NA,Finally,1376,1384 2976997,aaps j,2010,NA,Finally,851,859 2976990,aaps j,2010,NA,Significantly,460,474 2751391,aaps j,2008,NA,Clearly,712,720 2751391,aaps j,2008,NA,Importantly,1175,1187 3385822,aaps j,2012,NA,Additionally,798,811
There will of course be false positives – words ending with “ly,” that are not adverbs. Some of these include: the month of July, the country of Italy, surnames such as Whitely, medical conditions such as renomegaly and typographical errors such as “Findingsinitially“. These examples are uncommon and I just ignore them where they occur. There will also be sentence adverbs that do not include the comma but for now, I’m restricting the analysis to the “more dramatic” form, with commas.
3. Clean up the mess
Skip to the last sentence in this section if you want to avoid the tedious details of data cleaning.
You’ll note that in the Ruby code, extracted text was passed through chomp to remove end of line characters. This was not the case originally, since I was not expecting to find line breaks in the tag contents. However, it’s always good to check that the lines in your CSV file contain the expected number of fields (7):
which(count.fields("data/adverbs.csv", sep = ",") != 7)
[1] 55294 55295 55309 55310 55311 55312 55313 55314 55332 55333 55410 55411
[13] 55463 55464 55523 55524 55525 55526 55665 55666 55707 55708 56183 56184
[25] 56200 56201 56263 56264 57923 57924 57925 57926 57927 57928 57929 57930
[37] 57931 57932 57933 57934 57935 57936 57937 57938 57939 57940 57941 57942
[49] 57943 57944 57945 57946 57947 57948 57949 57950
Oh dear. Closer inspection indicates that we are looking at 28 pairs of adjacent lines. A quick look at the first 2 such lines in the CSV file:
tail -n+55294 adverbs.csv | head -2 540057,Nucleic Acids Res,2004 ,2005,Alternatively,946,960
Seems like our 7 fields are being broken by an unexpected new line. Surely, PubMed Central, you would not allow a line break in the pub-date[@pub-type='epub']/year field? Let’s look at PMC 540057:
<pub-date pub-type="epub">
<day>17</day>
<month>12</month>
<year>2004
</year>
You would. OK then.
Rather than run the code again, I wrote an ugly fix in R. It identifies the offending lines and pastes them back together. When that’s done, it figures out unique occurrences of sentence adverbs by assuming that additional records with the same PMC uid, adverb and start/end position in an abstract are duplicates. That last step is required because there are files with duplicate contents in the PMC dataset; something that I only discovered when summing adverbs per PMC uid and realising that some occur twice.
Ultimately we end up with a CSV file, adverbs.uniq.csv, containing 91 618 unique and cleaned records.
4. Analysis
R code for analysing the adverbs is in the file adverbs.R. It contains 4 functions: for counting adverbs, plotting the top 20 as a bar plot, visualizing the top 100 as a word cloud and examining adverb occurrence by journal.
4.1 Top 20 adverbs
Let’s start with a basic “top N” list. Plot on the left, word cloud on the right.
It seems that the most popular use of the sentence adverb is to draw a close to the proceedings, with finally. The next most common uses: to indicate further points of interest (additionally), label results as interesting (interestingly) or important (importantly) and show that the authors are up to date with their reading (recently).
Findings are frequently surprising, but more often unfortunate than remarkable.
Altogether, there are 710 unique words in the adverb column from 91 618 records. Note that this includes the false positives mentioned previously, including a number of typographical errors. For example, we find both phylogenitically and phylogentically, in addition to the correct term, phylogenetically (25 occurrences). Similarly, (see what I did there?) both intriguinly and intringuingly are present, as well as the correct intriguingly (648 occurrences).
4.2 Top 20 opening adverbs
If you’re anything like me, you have sat in front of a blank screen searching for that brilliant opening sentence for an article…and then typed:
“Recently,”
What inspiration did the authors of PMC find?
Same code, but applied to the subset of adverbs where match start = 0, i.e. the opening sentence of the abstract.
Looks like I’m not the only one who can’t do any better than recently. Authors also open by referring to previous work or assessing the current state of play.
4.3 The bad and the ugly
It’s possible to create all manner of adverbs by sticking -ly on the ends of things. This is rarely a good idea.
You might imagine that very long adverbs stand a good chance of being ugly. Let’s find the longest:
longest <- subset(adverbs.uniq, nchar(adverbs.uniq$adv) == max(nchar(adverbs.uniq$adv))) longest$adv # [1] "Electronmicroscopically"
You’d be correct.
You might also imagine that rarely-used adverbs stand a good chance of being ugly:
rarest <- subset(adverbs.freq, Freq == 1)
270 sentence adverbs appear only once in the dataset, so we’ll leave that as an “exercise for the reader”. Suffice to say that some of them include: endosonographically, ethnopharmacologically, ophthalmoscopically and tinctorially.
4.4 Adverbs by journal
It would be interesting to know whether adverbs are over-represented in particular journals. Simply counting the adverb by journal is no good, since some journals have many more articles in PMC than others. PLoS ONE, for example, accounts for almost 20% of all sentence adverb records:
head(jour[ order(jour$Freq, decreasing = T),]) # Var1 Freq # 2036 plos one 18285 # 1359 j cell biol 3053 # 1428 j exp med 2892 # 1897 nucleic acids res 2531 # 2033 plos genet 2076 # 2037 plos pathog 2043
Incidentally, this calculation is why we converted journal abbreviations to lower-case. PLoS ONE is also listed as PLoS One in PMC, but we want to count those names as one journal, hence plos one.
So, we need some sort of adjustment for total articles. I’ve gone with “occurrences per 100 adverbs.” That is: for every 100 sentence adverbs found in abstracts from a journal, how many occurrences of a particular adverb do we see? Furthermore, I’ve applied this metric to only those journals that have 100 or more sentence adverbs in the PMC dataset.
Let’s start with the surprising index (column a in this and subsequent tables).
surprising <- advIndex("Surprisingly", adverbs.jour, jour)
head(surprising[, 2:4], 10)
# Var2 Freq a
# 1440517 plos biol 94 11.032864
# 1443357 plos genet 191 9.200385
# 1521457 sci rep 26 8.965517
# 1312007 nature 24 8.695652
# 1302777 nat commun 9 8.411215
# 964817 j cell biol 250 8.188667
# 208667 bmc biol 12 7.500000
# 1446197 plos pathog 151 7.391092
# 1013807 j exp med 194 6.708160
# 940677 j biol chem 29 6.487696
The message seems clear: go with a Nature or specialist PLoS journal if your results are surprising.
How about interesting?
head(interesting[, 2:4], 10) # Var2 Freq a # 225387 bmc immunol 32 20.91503 # 945327 j biomed sci 19 19.00000 # 1010647 j exp bot 40 18.26484 # 1278317 mol neurodegener 22 18.18182 # 1616277 virol j 82 18.14159 # 1262697 mol cancer 81 17.96009 # 806167 int j biol sci 23 17.69231 # 1435937 plant physiol 19 17.27273 # 233907 bmc microbiol 83 16.50099 # 1280447 mol pain 26 16.04938
No clear winner there. Anyone for remarkable results?
head(remarkable[, 2:4], 10) # Var2 Freq a # 1311933 nature 24 8.695652 # 1440443 plos biol 36 4.225352 # 686423 genome biol 15 4.000000 # 687133 genome biol evol 5 3.937008 # 1284243 mol syst biol 9 3.422053 # 1502923 retrovirology 10 3.246753 # 1443283 plos genet 67 3.227360 # 1521383 sci rep 8 2.758621 # 740383 hum mol genet 3 2.702703 # 545133 embo mol med 3 2.631579
Save your most remarkable results for submission to Nature.
Finally, what if it’s all a bit unfortunate?
# Var2 Freq a # 1444113 plos med 106 8.870293 # 168953 bioinformatics 14 7.329843 # 1600313 trials 7 6.930693 # 210133 bmc biotechnol 12 6.451613 # 1072783 j med case reports 12 6.030151 # 208003 bmc bioinformatics 74 5.501859 # 252023 bmc syst biol 17 5.396825 # 1444823 plos negl trop dis 34 5.082212 # 1610963 vasc health risk manag 5 4.901961 # 186703 biomed eng online 5 4.761905
I’m a little surprised that bioinformatics features so prominently. What could be so unfortunate? The results of applying your method to real data, as opposed to simulated or training data?
4.5 Most sentence adverbs in an abstract
Who got a bit carried away with the sentence adverbs? Easy to find out by counting up the PMC uids:
pmc <- as.data.frame(table(adverbs.uniq$pmc)) pmc <- pmc[ order(pmc$Freq, decreasing = T),] head(pmc) # Var1 Freq # 11769 2214797 7 # 27434 2873921 7 # 39930 3130555 7 # 42598 3173291 7 # 49235 3280967 7 # 13 17814 6
There are five abstracts that contain 7 sentence adverbs. Going back to the PMC website, we see that these are:
Summary
As usual, this analysis is intended to be a bit of fun. Next time you’re writing that article though, ask yourself: is that sentence enhanced by the sentence adverb? Or are you simply following convention?
“Open”: motivation versus definition [What You're Doing Is Rather Desperate] 2013-07-10 18:00
Tweet length: 140 characters. Quote + URL that I wanted to tweet: 160 characters. Solution: brief blog post.
the probability that people who can help each other can be connected has risen to the point that for many types of problem that they actually are
Please read the rest of Cameron’s thoughts on motivations for openness in research: Open is a state of mind.
-omics in 2013 [What You're Doing Is Rather Desperate] 2013-06-25 01:51
Just how many (bad) -omics are there anyway? Let’s find out.
Update: code and data now at Github
1. Get the raw data
It would be nice if we could search PubMed for titles containing all -omics:
*omics[TITL]
However, we cannot since leading wildcards don’t work in PubMed search. So let’s just grab all articles from 2013:
2013[PDAT]
and save them in a format which includes titles. I went with “Send to…File”, “Format…CSV”, which returns 575 068 records in pubmed_result.csv, around 227 MB in size.
2. Extract the -omics
Titles are in column 1 and we only want the -omics, so:
cut -f1 -d "," pubmed_result.csv | grep -ioP "(\w+omics)" > omics.txt wc -l omics.txt # 1763 omics.txt
Note: grep changed so the following now works. Note: this approach will miss a very few cases where omics is preceded by a hyphen. That included the classic stain-omics..
It also ignores the standalone term “-omics”, which is used quite often
Of course, this results in some “false positives” (e.g. economics). Curation would be required to detect the “true -omics”.
3. Visualize
Time to break out the R. The top 20 -omics in 2013 and the less popular:
library(ggplot2)
omics <- readLines("omics.txt")
omics <- tolower(omics)
omics.freq <- as.data.frame(table(omics))
omics.freq <- omics.freq[ order(omics.freq$Freq, decreasing = T),]
omics.freq$omics <- factor(omics.freq$omics, levels = omics.freq$omics)
ggplot(head(omics.freq, 20)) + geom_bar(aes(omics, Freq), stat = "identity", fill = "darkblue") +
theme_bw() + coord_flip()
# and the less popular
subset(omics.freq, Freq == 1)
|
On the right, the top 20. Click for a larger version of the graphic. Top of the list so far for 2013 is proteomics, followed by genomics and metabolomics.
Listed below, those -omics found only once in titles from 2013. Some shockers, I think you’ll agree (paging Jonathan Eisen).
aquaphotomics 1
biointeractomics 1
calciomics 1
cholanomics 1
cytogenomics 1
cytokinomics 1
econogenomics 1
glcnacomics 1
glycosaminoglycanomics 1
interactomics 1
ionomics 1
macroeconomics 1
materiomics 1
metalloproteomics 1
metaproteogenomics 1
microbiomics 1
microeconomics 1
microgenomics 1
microproteomics 1
miromics 1
mitoproteomics 1
mobilomics 1
modomics 1
morphomics 1
museomics 1
neuromics 1
neuropeptidomics 1
nitroproteomics 1
nutrimetabonomics 1
oncogenomics 1
orthoproteomics 1
pangenomics 1
petroleomics 1
pharmacometabolomics 1
pharmacoproteomics 1
phylotranscriptomics 1
phytomics 1
postgenomics 1
pyteomics 1
radiogenomics 1
rehabilomics 1
retrophylogenomics 1
romics 1
secretomics 1
sensomics 1
speleogenomics 1
surfaceomics 1
surfomics 1
toxicometabolomics 1
vaccinomics 1
variomics 1
|
Never heard of romics? That’s OK. It’s a surname.
No-one cares about your bioinformatics software [What You're Doing Is Rather Desperate] 2013-06-23 17:36
Here’s a tip. When you write an article about your software, the title of which indicates that open-source is important:
but you then:
Don’t be surprised when no-one uses your software.
Or is the publication more important to you than the product?
Snippets: guts, cancers, statistics [What You're Doing Is Rather Desperate] 2013-06-17 19:35
File under “interesting articles that I don’t have time to write about at length.”
Long ago, before metagenomics and NGS, I did a little work on detection of Archaea in human microbiomes. There’s a blog post in the pipeline about that but until then, enjoy this article in PLoS ONE.
This article is getting a lot of attention on Twitter this week. Brief summary: cancer cells are really messed up in all sorts of ways, most of which are not causal with respect to the cancer. Anyone who has ever looked at microarray data knows that it’s not uncommon for 50% or more of genes to show differential expression in a cancer/normal comparison, so this is hardly a new concept. I think we need to move away from ever-more detailed characterizations of the ways in which cancer cells are “messed up.” We know that they are and that doesn’t provide much insight, in my opinion.
Interesting post by Jeff Leek, summarized very well by its title. It points out that many more people are now interested in data analysis, many of them are not trained professionally as statisticians (I’m in this category myself) and we need to recognize and plan for that.
Bonus post doing the rounds of social media: Using Metadata to Find Paul Revere. Social network analysis, 18th-century style. Amusing, informative and topical.
Using the Ensembl Variant Effect Predictor with your 23andme data [What You're Doing Is Rather Desperate] 2013-06-04 00:28
I subscribe to the Ensembl blog and found, in my feed reader this morning, a post which linked to the Variant Effect Predictor (VEP). The original blog post, strangely, has disappeared.
Not to worry: so, the VEP takes genotyping data in one of several formats, compares it with the Ensembl variation + core databases and returns a summary of how the variants affect transcripts and regulatory regions. My first thought – can I apply this to my own 23andme data?
1. Convert 23andme data to VCF
If you download your raw data from 23andme, it looks something like this (ignoring comment lines):
rs4477212 1 82154 AA rs3094315 1 752566 AA rs3131972 1 752721 GG rs12562034 1 768448 GG rs12124819 1 776546 AA
The rest of this post assumes that your raw data are saved in a file named mySNPs.txt; note that the original file name of your download will be something like genome_YOUR_NAME_Full_TIMESTAMP.txt.zip.
VEP will accept several file formats, including VCF (variant call format). At Github, I discovered a tool named 23andme2vcf to perform the conversion. My first attempt failed:
perl 23andme2vcf.pl mySNPs.txt mySNPs.vcf # raw data file and reference file are out of sync at ./23andme2vcf.pl line 154, line 4.
Digging around the Github issues page, I noted that this error has occurred before; the issue was closed when the reference file supplied with 23andme2vcf was updated.
A quick line count reveals the problem: more SNPs in my 23andme data (V3 platform) than the reference file.
gunzip -c 23andme_hg19ref_20121017.txt.gz | wc -l # 960613 grep -cv "^#" mySNPs.txt # 991786
Easiest fix: read both into R, discard those SNPs not found in the reference file and write back out to a new tab-delimited file. To avoid discarding SNPs, you’ll have to figure out how to create the reference file; perhaps the issue will be fixed in a future update.
ref <- read.table("23andme_hg19ref_20121017.txt.gz", header = F, stringsAsFactors = F)
snp <- read.table("mySNPs.txt", header = F, stringsAsFactors = F)
snp <- subset(snp, V1 %in% ref$V3)
write.table(snp, "mynewSNPs.txt", quote = F, col.names = F, row.names = F, sep = "\t")
Now the conversion works as expected:
perl 23andme2vcf.pl mynewSNPs.txt mynewSNPs.vcf
Note that the script does not yet handle insertions or deletions. However, these are not well-defined in the 23andme data file in any case.
2. Install VEP locally
This is all as described in the VEP script documentation. Click the “download latest version” link, navigate to wherever you downloaded it and then:
tar zxvf variant_effect_predictor.tar.gz cd variant_effect_predictor perl INSTALL.pl
Follow the prompts. You will want to install the core and VEP files for Homo sapiens, the latest release being 71. Note that VEP files are a large download, currently ~ 2.5 GB, which will take some time.
3. Run VEP
From the variant_effect_predictor directory, as simple as:
perl variant_effect_predictor.pl --cache -i ~/path/to/mynewSNPs.vcf -o ~/path/to/myVEP.txt
In addition to the delimited text output (mine is around 163 MB in size), the script generates a rather nice HTML summary with some statistics. It’s also easy to read the output from VEP back into R. See plot, right, for a summary of the VEP Consequence column.
library(ggplot2)
vep <- read.table("vep.txt", header = F, stringsAsFactors = F, sep = "\t")
colnames(vep) <- c("Uploaded_variation", "Location", "Allele", "Gene", "Feature", "Feature_type",
"Consequence", "cDNA_position", "CDS_position", "Protein_position", "Amino_acids",
"Codons", "Existing_variation", "Extra")
cons <- as.data.frame(table(vep$Consequence))
cons <- cons[sort.list(cons$Freq, decreasing = T),]
cons$Var1 <- factor(cons$Var1, levels = cons$Var1)
ggplot(cons) + geom_bar(aes(Var1, log10(Freq)), stat = "identity") + coord_flip() + theme_bw()
It’s a lot of output to trawl through and summarize; I’ll post on the topic again if I discover anything interesting.
Unconference on the Future of Statistics (Live Stream) #futureofstats [Simply Statistics] 2013-10-30 11:36
The Unconference on the Future of Statistics will begin at 12pm EDT today. Watch the live stream here.
How to participate in #futureofstats Unconference [Simply Statistics] 2013-10-29 09:52
Tomorrow is the Unconference on the Future of Statistics from 12PM-1PM EDT. There are two ways that you can get in the game:
Tukey Talks Turkey #futureofstats [Simply Statistics] 2013-10-29 09:38
I've been digging up old "future of statistics" writings from the past in anticipation of our Unconference on the Future of Statistics this Wednesday 12-1pm EDT. Last week I mentioned Daryl Pregibon's experience trying to build statistical expertise into software. One classic is "The Future of Data Analysis" by John Tukey published in the Annals of Mathematical Statistics in 1962.
Perhaps the most surprising aspect of this paper is how relevant it remains today. I think perhaps with just a few small revisions it could easily be published in a journal today and few people would find it out of place.
In Section 3 titled "How can new data analysis be initiated?" he describes directions in which statisticians should go to grow the field of data analysis. But the advice itself is quite general and probably should be heeded by any junior statistician just starting out in research.
How is novelty most likely to begin and grow? Not through work on familiar problems, in terms of familiar frameworks, and starting with the results of applying familiar processes to the observations. Some or all of these familiar constraints must be given up in each piece of work which may contribute novelty.
Tukey's article serves as a coherent and comprehensive roadmap for the development of data analysis as a field. He suggests that we should study how people analyze data and uncover "what works" and what doesn't. However, he appears to draw the line at suggesting that such study should result in a single way of analyzing a given type of data. Rather, statisticians should maintain some flexibility in modeling and analysis. I personally think the reality should be somewhere the middle. Too much flexibility can lead to problems, but rigidity is not the solution.
It is interesting, from my perspective, that given how clear and coherent Tukey's roadmap was in 1962, how much of it was essentially ignored. In fact, the field pretty much went the other direction towards more mathematical elegance (I'm guessing Tukey sensed this would happen). His article is uncomfortable to read, because it's full of problems that arise in real data that are difficult to handle with standard approaches. He has an uncanny ability to make up methods that look totally bizarre on first glance but are totally reasonable after some thought.
I honestly can't think of a better way to end this post than to quote Tukey himself.
The future of data analysis can involve great progress, the overcoming of real difficulties, and the provision of a great service to all fields of science and technology. Will it? That remains to us, to our willingness to take up the rocky road of real problems in preference to the smooth road of unreal assumptions, arbitrary criteria, and abstract results without real attachments. Who is for the challenge?
Read the paper. And then come join us at 12pm EDT tomorrow.
Simply Statistics Future of Statistics Speakers - Two Truths, One Lie #futureofstats [Simply Statistics] 2013-10-28 10:28
Our online conference live-streamed on Youtube is going to happen on October 30th 12PM-1PM Baltimore (UTC-4:00) time. You can find more information here or sign up for email alerts here. I get bored with the usual speaker bios at conferences so I am turning our speaker bios into a game. Below you will find three bullet pointed items of interest about each of our speakers. Two of them are truths and one is a lie. See if you can spot the lies and sign up for the unconference!
Sunday data/statistics link roundup (10/27/13) [Simply Statistics] 2013-10-27 13:16
(Back to) The Future of Statistical Software #futureofstats [Simply Statistics] 2013-10-25 08:24
In anticipation of the upcoming Unconference on the Future of Statistics next Wednesday at 12-1pm EDT, I thought I'd dig up what people in the past had said about the future so we can see how things turned out. In doing this I came across an old National Academy of Sciences report from 1991 on the Future of Statistical Software. This was a panel discussion hosted by the National Research Council and summarized in this volume. I believe you can download the entire volume as a PDF for free from the NAS web site.
The entire volume is a delight to read but I was particularly struck by Daryl Pregibon's presentation on "Incorporating Statistical Expertise into Data Analysis Software" (starting on p. 51). Pregibon describes his (unfortunate) experience trying to develop statistical software which has the ability to incorporate expert knowledge into data analysis. In his description of his goals, it's clear in retrospect that he was incredibly ambitious to attempt to build a kind of general-purpose statistical analysis machine. In particular, it was not clear how to incorporate subject matter information.
[T]he major factor limiting the number of people using these tools was the recognition that (subject matter) context was hard to ignore and even harder to incorporate into software than the statistical methodology itself. Just how much context is required in an analysis? When is it used? How is it used? The problems in thoughtfully integrating context into software seemed overwhelming.
Pregibon skirted the problem of integrating subject matter context into statistical software.
I am not talking about integrating context into software. That is ultimately going to be important, but it cannot be done yet. The expertise of concern here is that of carrying out the plan, the sequence of steps used once the decision has been made to do, say, a regression analysis or a one-way analysis of variance. Probably the most interesting things statisticians do take place before that.
Statisticians (and many others) tend to focus on the application of the "real" statistical method--the regression model, lasso shrinkage, or support vector machine. But as much painful experience in a variety of fields has demonstrated, much what happens before the application of the key model is as important, or even more important.
Pregibon makes an important point that although statisticians are generally resistant to incorporating their own expertise into software, they have no problem writing textbooks about the same topic. I've observed the same attitude when I talk about evidence-based data analysis. If I were to guess, the problem is that textbooks are still to a certain extent abstract, while software is 100% concrete.
Initial efforts to incorporate statistical expertise into software were aimed at helping inexperienced users navigate through the statistical software jungle that had been created…. Not surprisingly, such ideas were not enthusiastically embraced by the statistics community. Few of the criticisms were legitimate, as most were concerned with the impossibility of automating the “art” of data analysis. Statisticians seemed to be making a distinction between providing statistical expertise in textbooks as opposed to via software. [emphasis added]
In short, Pregibon wanted to move data analysis from an art to a science, more than 20 years ago! He stressed that data analysis, at that point in time, was not considered a process worth studying. I found the following paragraph interesting and worth considering in now, over 20 years later. He talks about the reasons for incorporating statistical expertise into software.
The third [reason] is to study the data analysis process itself, and that is my motivating interest. Throughout American or even global industry, there is much advocacy of statistical process control and of understanding processes. Statisticians have a process they espouse but do not know anything about. It is the process of putting together many tiny pieces, the process called data analysis, and is not really understood. Encoding these pieces provides a platform from which to study this process that was invented to tell people what to do, and about which little is known. [emphasis added]
I believe we have come quite far since 1991, but I don't think we no much more about the process of data analysis, especially in newer areas that involve newer data. The reason is because the field has not put much effort into studying the whole data analysis process. I think there is still a resistance to studying this process, in part because it involves "stooping" to analyze data and in part because it is difficult to model with mathematics. In his presentation, Pregibon suggests that resampling methods like the bootstrap might allow us to skirt the mathematical difficulties in studying data analysis processes.
One interesting lesson Pregibon relates during the development of REX, an early system that failed, involves the difference between the end-goals of statisticians and non-statisticians:
Several things were learned from the work on REX. The first was that statisticians wanted more control. There were no users, rather merely statisticians looking over my shoulder to see how it was working. Automatically, people reacted negatively. They would not have done it that way. In contrast, non-statisticians to whom it was shown loved it. They wanted less control. In fact they did not want the system--they wanted answers.
The Leek group guide to reviewing scientific papers [Simply Statistics] 2013-10-23 11:14
There has been a lot of discussion of peer review on this blog and elsewhere. One thing I realized is that no one ever formally taught me the point of peer review or how to write a review.
Like a lot of other people, I have been frustrated by the peer review process. I also now frequently turn to my students to perform supervised peer review of papers, both for their education and because I can't handle the large number of peer review requests I get on my own.
So I wrote this guide on how to write a review of a scientific paper on Github. Last time I did this with R packages a bunch of people contributed to make the guide better. I hope that the same thing will happen this time.
Blog posts that impact real science - software review and GTEX [Simply Statistics] 2013-10-22 11:53
There was a flurry of activity on social media yesterday surrounding a blog post by Lior Pachter. He was speaking about the GTEX project - a large NIH funded project that has the goal of understanding expression variation within and among human beings. The project has measured gene expression in multiple tissues of over 900 individuals.
In the post, the author claims that the GTEX project is "throwing away" 90% of its data. The basis for this claim is a simulation study using the parameters from one of the author's papers. The claim of 90% is based on the fact that increasing the number of mRNA fragments leads to increasing correlation in abundance measurements in the simulation study. In order to get the same Spearman correlation as other methodologies have at 10M fragments, the software being used by GTEX needs 100M fragments.
This post and the associated furor raises three issues:
The first point is obvious; the post was rapidly disseminated and elicited responses from the leaders of the GTEX project. Interestingly, I think the authors got an early view of the criticisms they would face from reviewers through the blog post. The short term criticism is probably not fun to deal with but it might save them time later.
I think the criticism about using software that has not been fully vetted through the publication/peer review process is an important one. For such a large scale project, you'd like to see the primary analysis being done with "community approved" software. The reason is that we just don't know if it is better or worse because no one published a study on the software. It would be interesting to see how the bottom up approach would have faired here. The good news for GTEX here is that for future papers they will either get out a more comprehensive comparison or they will switch software - either of which will improve their work.
Regarding point 2, Pachter did a "back of the envelope" calculation that suggested the Flux software wasn't performing well. These back of the envelope calculations are very important - if you can't solve the easy case, how can you expect to solve the hard case. Lost in all of the publicity about the 90% number is that Pachter's blog post hasn't been vetted, either. Here are a few questions that immediately jumped to my mind when reading the blog post:
Whenever a scientist sees a claim as huge as "throwing away 90% of the data" they should be skeptical. This is particularly true in genomics, where huge effects are often due to bugs or artifacts. So in general, it is important that we apply the same level of scrutiny to extreme critiques as we do to extreme claims.
My guess is ultimately, the 90% number may end up being an overestimate of how bad the problem is. On the other hand, I think it was hugely useful for Pachter to point out the potential issue and give GTEX the chance to respond. If nothing else, it points out (1) the danger of using unpublished methods when good published alternatives exist and (2) that science moves faster in the era of blog posts and social media.
Disclaimers: I work on RNA-seq analysis although I'm not an author on any of the methods being considered. I have spoken at a GTEX meeting, but am not involved in the analysis of the data. Most importantly, I have not analyzed any data and am in no position to make claims about any of the software in question. I'm just making observations about the sociology of this interaction.
PubMed commons is launching [Simply Statistics] 2013-10-22 11:00
Why are the best relievers not used when they are most needed? [Simply Statistics] 2013-10-21 10:00
During Saturday's ALCS game 6 the Red Sox's manager John Farrell took out his starter in the 6th inning. They were leading by 1, but had runners on first and second with no outs. This is a hard situation to get out of without giving up a run. The chances of scoring with an average pitcher are about 64%. I am sure that with a top of the line pitcher, like Koji Uehara, this number goes down substantially. So what does a typical manager do in this situation? Because managers like to save their better relievers for the end, and it's only the 6th inning, they will bring in a mediocre one instead. This is what Farrell did and 2 batters latter the score was 2-1 Tigers. To really understand why this is bad move, the chances of a mediocre pitcher giving up runs when starting an inning is about 28%. So why not bring in your best reliever when the game is actually on the line? Here is an article by John Dewan with a good in -depth discussion. Note that the Red Sox won the game 5-2 and Koji Uehara was brought in the ninth inning to get 3 outs with the bases empty and a 3 run lead.
Streamline Your Mechanical Turk Workflow with MTurkR [Solomon Messing] 2013-06-24 12:20
I’ve been using Thomas Leeper‘s MTurkR package to administer my most recent Mechanical Turk study—an extension of work on representative-constituent communication claiming credit for pork benefits, with Justin Grimmer and Sean Westwood. MTurkR is excellent, making it quick and easy to:
In this post I’ll walk you through the set-up process and a couple of basic examples. But first a few words to motivate your interest in Mechanical Turk.
Mechanical Turk has proven to be a boon for social science research. I discussed how it’s a great way attain data based on human judgements (e.g., content analysis, coding images, etc.) in my last post on creating labels for supervised text classification.
It’s also a great source of experimental subjects in a field often plagued by small samples, which have lower power and higher false discovery rates (especially when researchers exploit “undisclosed flexibility” in their design and analysis). Mechanical Turk provides a large, geographically and demographically diverse sample of participants, freeing researchers from confines of undergraduate samples, which can respond differently than other key populations.
Mechanical Turk also provides a much lower-cost experimental platform. Compared to a national survey research firm, the cost per subject is dramatically lower, and most survey firms simply do not allow the same design flexibility, generally limiting you to question-wording manipulations. Mechanical Turk is also cheaper than running a lab study in terms of the researcher’s time, or that of her graduate students. This is true even for web studies—just tracking undergraduate participants and figuring out who should get course credit is a mind-numbing, yet error-prone and so frustrating process. Mechanical Turk makes it easy for the experimenter to manage data collection, perform validation checks, and quickly disburse payment to subjects who actually participated. I’ll show you how to streamline this process to automate as much as possible in the next section.
But are results from Mechanical Turk valid and will I be able to publish on Mturk data? Evidence that the answer is “yes” accumulates. Berinksy, Huber, and Lenz (2012) published a validation study in Political Analysis that replicates important published experimental work using the platform. Furthermore, Justin Grimmer, Sean Westwood and I published a study in the American Political Science Review on how people respond when congressional representatives announce political pork in which we provide extensive Mechanical Turk validation in the online appendix. We provide demographic comparisons to the U.S. Census, show the regional distribution of Turkers by state and congressional district, and show that correlations between political variables are about the same as in national survey samples. Sean and I also published a piece in Communication Research on how social cues drive the news we consume which among other things replicates past findings on partisans’ preferences for news with a partisan slant.
But are Turkers paying enough attention to actually process my stimuli? It’s hard to know for sure, but there are an array of questions one can deploy that are designed to make sure Turkers are paying attention. When my group runs Mturk studies, we typically ask a few no-brainer questions to qualify at the beginning of any task (e.g., 5 + 7 = 13, True or False?), then burry some specific instructions in a paragraph of text toward the end (e.g., bla bla bla, write “I love Mechanical Turk” in the box asking if you have any comments).
But we are paying Turkers, doesn’t that make them more inclined toward demand effects? Well, compared to what? Compared to a student who needs class credit or a paid subject? On the contrary, running a study on the web ensures that researchers in a lab do not provide non-verbal cues (of which they are unaware) that can give rise to demand effects. Of course, in any experimental setting it’s important to think carefully about potential demand effects, and especially so when deploying noticeable/obvious manipulations, and or when you have a within-subject design such that respondents observe multiple conditions.
Set-Up
Install the most recent version of MTurkR from Tom’s github repo. You can use Hadley’s excellent devtools package to install it:
# install.packages("devtools")
library(devtools)
install_github(repo="MTurkR",
username = "leeper")
If you don’t have a Mechanical Turk account, get one. Next you want to set up your account, fund it, etc., there are a lot of good walkthroughs just google it if you have trouble.
Also, set up your sandbox so you can test things before you actually spend real money.
You’ll also need to get your AWS Access Key ID and AWS Secret Access Key to get access to the API. Click on “Show” under “Secret Access Key” to actually see it.
I believe you can use the MTurkR package to design a questionnaire and handle randomization, but that’s beyond the scope of this post—for now just create a project as you normally would via the GUI.
First, make sure your credentials work correctly and check your account balance (replace the dummy credentials with your own):
require("MTurkR")
credentials(c("AKIAXFL6UDOMAXXIHBQZ","oKPPL/ySX8M7RIXquzUcuyAZ8EpksZXmuHLSAZym"))
AccountBalance()
Great, now R is talking to the API. Now you can actually create HITs. Let’s start in your sandbox. Create a project in your sandbox, or just copy a project you’ve already created—copy the html in the design layout screen from your production account to your sandbox account.
Now you need to know the HIT layout id. Make sure you are in the “Create” tab in the requester interface, and click on your Project Name. This window will pop up:
Copy and paste the Layout ID in your R script, you’ll use it in a second. With the Layout ID in hand, you can create HITs based on this layout. Let’s create a HIT in our sandbox first. We’ll first set the qualifications that Mechanical Turkers must meet, then tell the API to create a new HIT in the sandbox with 1200 assignments for $.50 each. Modify the parameters below to fit your needs.
# First set qualifications # ListQualificationTypes() to see different qual types qualReqs = paste( # Set Location to US only GenerateQualificationRequirement( "Location","==","US"), # Worker_PercentAssignmentsApproved GenerateQualificationRequirement( "000000000000000000L0", ">", "80", qual.number=2), # Un-comment after sandbox test # Worker_NumberHITsApproved # GenerateQualificationRequirement( # "00000000000000000040", ">", "100", # qual.number=3), sep="" ) # Create new batch of hits: newHIT = CreateHIT( # layoutid in sandbox: hitlayoutid="22P2J1LY58B74P14WC6KKD16YGOR6N", sandbox=T, # layoutid in production: # hitlayoutid="2C9X7H57DZKPHWJIU98DS25L8N41BW", annotation = "HET Experiment with Pre-Screen", assignments = "1200", title="Rate this hypothetical representative", description="It's easy, just rate this hypothetical representative on how well she delivers funds to his district", reward=".50", duration=seconds(hours=4), expiration=seconds(days=7), keywords="survey, question, answers, research, politics, opinion", auto.approval.delay=seconds(days=15), qual.reqs=qualReqs )
To check to make sure everything worked as intended, go to your worker sandbox and search for the HIT you just created. See it? Great.
Here’s how to check on the status of your HIT:
# Get HITId (record result below) newHIT$HITId # "2C2CJ011K274LPOO4SX1EN488TRCAG" HITStatus(hit="2C2CJ011K274LPOO4SX1EN488TRCAG")
And now here’s where the really awesome and time-saving bit comes in, downloading the most recent results. If you don’t use the API, you have to do this manually from the website GUI, which is a pain. Here’s the code:
review = GetAssignments(hit="2C2CJ011K274LPOO4SX1EN488TRCAG", status="Submitted", return.all=T)
Now you probably want to actually run your study with real subjects. You can copy your project design HTML back to the production site and repeat the above for production when you are ready to launch your HIT.
Often, Turkers will notice something about your study and point it out, hoping that it will prove useful and you will grant them a bonus. If they mention something helpful, grant them a bonus! Here’s how (replace dummy workerid with the id for the worker to whom you wish to grant a bonus):
## Grant bonus to worker who provided helpful comment: bonus assignments=review$AssignmentId[review$WorkerId=="A2VDVPRPXV3N59"], amounts="1.00", reasons="Thanks for the feedback!")
You’ll also save time when you go to validate and approve HITs, which MTurkR allows you to do from R. My group usually uses Qualtrics for questionnaires that accompany our studies where we build in the attention check described above. Here’s how to automate the attention check and approve HITs for those who passed:
svdat = read.csv(unzip("data/Het_Survey_Experiment.zip"), skip=1, as.is=T)
approv = agrep("I love Mechanical Turk", max.distance=.3,
svdat$Any.other.comments.or.questions.)
svdat$Any.other.comments.or.questions.[approv]
correct = review$AssignmentId[
gsub(" ", "", review$confcode) %in% svdat$GUID[approv] ]
# To approve:
approve = approve(assignments=correct)
Note that “agrep()” is an approximate matching function which I use in case Turkers add quotes or other miscellaneous text that shouldn’t disqualify their work.
But that’s not all–suppose we want to check the average time per HIT (to make sure we are paying Turkers enough) and/or start analyzing our data—we can do this via the API rather than downloading things from the web with MTurkR.
# check average time: review = GetAssignments(hit="2C2CJ011K274LPOO4SX1EN488TRCAG", status="Approved", return.all=T) # Check distribution of time each Turker takes: quantile(review$SecondsOnHIT/60)
If we discover we aren’t paying Turkers enough in light of how long they spend on the HIT, which in addition to being morally wrong means that we will probably not collect a sufficient number of responses in a timely fashion, MTurkR allows us to quickly remedy the situation. We can expire the HIT, grant bonuses to those who completed the low-pay HIT, and relaunch. Here’s how:
# Low pay hits:
HITStatus(hit="2JV21O3W5XH0L74WWYBYKPLU3XABH0")
# Review remaining HITS and approve:
review = GetAssignments(hit="2JV21O3W5XH0L74WWYBYKPLU3XABH0",
status="Submitted", return.all=T)
correct = review$AssignmentId[ which( gsub(" ", "", review$confcode) %in% svdat$GUID[approv] ) ]
approve = approve(assignments=correct)
# Expire remaining:
ExpireHIT(hit="2JV21O3W5XH0L74WWYBYKPLU3XABH0")
approvedLowMoneyHits = GetAssignments(hit="2JV21O3W5XH0L74WWYBYKPLU3XABH0",
status="Approved", return.all=T)
# Grant bonus to workers who completed hit already:
bonus = GrantBonus(workers=approvedLowMoneyHits$WorkerId,
assignments=approvedLowMoneyHits$AssignmentId,
amounts="1.00",
reasons="Upping the pay for this HIT, thanks for completing it!")
We can now run the administrative side of our Mechanical Turk HITs from R directly. It’s a much more efficient workflow.
Generating Labels for Supervised Text Classification using CAT and R [Solomon Messing] 2013-02-04 09:36
The explosion in the availability of text has opened new opportunities to exploit text as data for research. As Justin Grimmer and Brandon Stewart discuss in the above paper, there are a number of approaches to reducing human text to data, with various levels of computational sophistication and human input required. In this post, I’ll explain how to use the Coding Analysis Toolkit (CAT) to help you collect human evaluations of documents, which is a necessary part of many text analyses, and especially so when you have a specific research question that entails precisely characterizing whether a particular document contains a particular type of content. CAT facilitates fast data entry and handles data management when you have multiple human coders. It’s default output can be tricky to deal with however, so I’ll also provide R code to extract useable data from CAT’s XML output, which should serve as a good into to data munging with XML to the uninitiated. I’ll also show you how to compute metrics that will help diagnose the reliability of your coding system, which entails using the melt and cast functionality in Hadley’s ‘reshape’ package to get the data in the right shape then feeding the results to the ‘irr’ package.
In future posts, I’ll explain how to use these labels to train various machine learning algorithms aka classification models to automatically classify documents in a large corpus. I’ll talk about how to extract features using R and the ‘tm’ package; the problem of classification in high-dimensional spaces (e.g., there are many many words in natural language) and how we can exploit the bias-variance tradeoff to get traction on this problem; the various models that are generally well suited for text classification like the lasso, elastic net, SVMs and random forests; the importance of properly tuning these models; and how to use cross-validation to avoid overfitting these models to your data. (For a preview check out my slides and R labs for my short course on Analyzing Text as Data that I presented at the Stanford Computational Social Science Workshop)
Social scientists have been trying to reduce the complexities and subjectivities of the human language to objective data for a long time, calling it “content analysis.” It is no easy task. If you decide to use human coders, I suggest you read Kim Neuendorf’s book and you can find some nice resources on her website that may prove helpful. You’d also do well to read Krippendoff’s eponymous classic.
If you are trying to characterize the type of things that occur or discover categories in your documents, it might make more sense to go with a fully computational approach, employing unsupervised machine learning methods that cluster documents based on word features (e.g. simple word counts, unigrams, or combinations thereof, N-grams). You can take a look at the Grimmer and Stewart paper above for more details on this approach, or check out the notes from lectures 5 – 7 from Justin’s course on the topic. If your are interested in additional computational approaches/features, have a look at Dan Jurafsky’s course From Language to Information.
But if you have a specific research question in mind, which entails precisely characterizing whether a particular document contains a particular type of content, you probably need people to read and classify each document according to a coding scheme. Of course, this can become expensive or impossible if you are dealing with a large corpus of documents. If so, you can first have people classify a sample of documents, which can then be used as labels to train supervised classifiers. Once you have a classifier that performs well, you can use it to classify the rest of the documents in your large data set. I have put together some slides on this process, along with an R lab with example code. Also of interest may be some slides I put together on acquiring and pre-processing text data from the web with R, along with computational labs on interacting with APIs and scraping/regex.
If instead you care about making generalizations about the proportion of documents in any given category (in your population of documents based on your sample), check out the R package ReadMe, which implements A Method of Automated Nonparametric Content Analysis for Social Science, and an industrial implementation of the method is offered at big data social media analytics company Crimson Hexagon. If you decide on this approach, you’ll still need human-labeled categories to start, so keep reading.
Before we get to CAT, I want to talk about human classification with Amazon’s Mechanical Turk, which is great when the classification task is simple and the documents/units of text to classify are short. Mturk is especially useful when the categorization task becomes mind-numbingly boring with repetition, because when one Turker burns out on your task and stops working, fresh Turkers can continue the work. Hence, the main advantage to Mturk is that you can get simple classification tasks done much more quickly than by relying upon research assistants/undergraduates/employees/etc—in fact I’ve used Mturk to classify thousands of short open responses to the question “What’s the most important issue facing the nation” into Gallup’s categories in a matter of hours.
Often overlooked are the nice interfaces that Mturk provides both to it’s requesters (e.g., you), which makes data management far easier than keeping track of excel spreadsheets/google docs edited by multiple coders, and to it’s workers, which translate to less mouse clicks, fatigue, and probably lower error rates. Panos Ipeirotis has some nice slides (best stuff in 30-50) + open source code to help ensure this kind of crowd-sourced data is of the highest quality.
But often the classification task is complicated and people need training to do it correctly and efficiently. I’d direct you to the various links above for guidance on building codebooks for complicated classification schemes and training coders. A great human-coder system is well-conceptualized, highly relevant to the corpus in question, and contains crystal clear instructions for your coders—providing flow charts and diagrams seems to be especially helpful. When Justin, Sean and I were implementing a coding protocol recently, we used this flow chart (from this latex file ) to compliment our actual codebook.
My experiences with Mechanical Turk spoiled me—all of the complexities of dealing with multiple coders entering data and managing that data were abstracted away by the system that Mturk has in place. What I’d done in the past—having analysts enter data in a Google Doc or worse, MS-Excel—was keystroke and mouse-click intensive, which meant it was time-consuming and error-prone for coders when they were entering data, and for me when I was merging/cleaning data from multiple coders.
CAT is the best solution I’ve come across yet. It’s interface isn’t aesthetically perfect, but it gets the job done well. It minimizes key strokes and requires no mouse clicks for data-entry, so you’ll see your coders work faster and probably happier. It maintains your data, alleviating the need to manage spreadsheets and concerns about your coders making errors due to transcribing codes to a spreadsheet. Because it also handles the back-end of things, there’s no need to maintain your own servers/sql database/etc. But it’s open-source so if you need to use your own servers, you can download the source and set it up yourself.

Head over to the CAT website and register for an account. Maybe poke around a bit on the website a bit, familiarize yourself with the menus. When you’re ready to upload a data set, go to Datasets –> Upload Raw Data Set. CAT wants a .zip file with all of your documents in individual .txt files.
If you have your text in a vector in R, say called text_vector, you can output each element to individual .txt files as follows:
for( i in 1:length(text_vector)){
capture.output(text_vector[i], file = paste("doc_number_", i, ".txt", sep="") )
}
Next put these files in a zip archive and upload to CAT. You can upload another file that specifies the coding scheme, but it’s probably easier just to make the coding scheme using the interface on the website later.
When you’re done, go to Datasets –> View Raw Datasets and click on the dataset you just uploaded. From this page, you can manage coders associated with the data sets. If you click on Manage Sub-Accounts, you can easily create new accounts for your coders. Add yourself and other coders to the dataset and be sure to click the “Set Chosen Coders” button when you’re done.
Next, implement your carefully constructed coding scheme. From the same “View Raw Dataset” page, click on “Add or modify codes in the dataset” (on the right under “Toolbox”). Add a sensical name for each code and enter a shortcut key—this will make it so your coders can just hit a button to code a document and move on to the next. When you’re done, hit finished.
I highly recommend you test out your coding scheme yourself. You’ll also probably want to consult your coders and iteratively tweak your codebook according to qualitative input from your coders (this is discussed in full in the content analysis links above).
Then set your coders loose on a hundred documents or so.
This process is a bit more complicated than it sounds. If you download data in CSV format, it comes out jagged (i.e., not rectangular), and hence it’s not immediately useful in R. The .CSV file becomes especially convoluted if you change your code labels, add coders, etc.
Better to just deal with the XML output. I’ll introduce data-munging/ETL with XML below. XML is like HTML, but tags describe data, not formatting. We’ll use those tags to create queries that return the data we want. XML is semi-structured data, with a general tree structure, rather than the rectangular structure that R likes. This tree structure saves space and is highly flexible, though it can be hard to work with initially. If you’ve never seen XML before, you’d do well to check out Jennifer Widom’s excellent lecture on XML in her Coursera course. XML is often used in various useful data APIs, which you can learn more about by checking out Sean Westwood’s short course on the topic.
To get the XML output for your project, from the “View Raw Dataset” page, select “Download Coded Text File (XML Format)” from the drop-down menu and then click on “Download Data” (on the right under “Toolbox”).
Here’s how to read in and clean the resulting file. You need to do this step because (1) R wants the encoding to be utf-8 but the CAT file says it’s utf-16, and (2) the XML package doesn’t like “�” strings (HTML notation for NULL), which frequently occur in the CAT output.
doc <- readLines("http://dl.dropbox.com/u/25710348/blog/sample.xml")
# check out what's in the file:
head(doc)
# Fix utf-8 issue:
doc <- gsub("utf-16", "utf-8", doc)
# Remove bad "�" characters:
grep("�", doc)
doc <- gsub("�", "", doc)
First a bit of background on this particular data. These represent a small subsample of documents that Justin Grimmer, Sean Westwood and I were putting together for a project looking at how congressional representatives claim credit for expenditures in their district. We were most concerned with identifying press releases that claimed credit for an expenditure in the district (CC_expenditure). But we also wanted to code items that explicitly criticized earmarks or advocated for earmark reform (Egregious); items that were speaking to local constituents—advertising constituent service that the candidate performed or community events the candidate attended to build name recognition (Adv_par_const); and items that were explicitly taking a national policy position or speaking to non-local audiences (Position_taking_other).
Have a look at the file above in an editor so you get a sense of its structure. The file first provides a lot of meta-data about the project, about each of the codes used, the coders, then the full text of each document. After the full text comes the actual data we want—how each item (paragraphId) was coded (codeId) and by which coder (coderId). It looks like this:
<codedDataItem>
<paragraphId>4334458</paragraphId>
<codeId>142061</codeId>
<coderId>4506</coderId>
</codedDataItem>
It’s the values inside each of the tags that we want. Here’s how we can get them: (1) parse the XML so R recognizes the tags and values properly using the XML package, and (2) extract those values and get them into a data frame for analysis using XPATH. (2) involves telling R to traverse the XML data and return the value in each of the paragraphId, codeId and coderId tags.
# Parse the XML
# uncomment the line below to install the XML package
# install.packages('XML')
library('XML')
doc <- xmlInternalTreeParse(doc, asText=T)
# That was easy, now for #2:
para = unlist(xpathApply(doc, "//paragraphId", xmlValue))
code = unlist(xpathApply(doc, "//codeId", xmlValue))
coderid = unlist(xpathApply(doc, "//coderId", xmlValue))
# Now put into a data frame:
alldat <- data.frame(para, coder=coderid, code)
That’s great, but if you want human-readable data, you need to do a few more things. Let’s pull each of the codeIds and codenames, then use that to map each of the codeIds in our data back to human-readable codes. We’ll do the same thing for our coders and give a number to each of the coding units (paragraphId).
# now map back to human readable values: # CODES codeids <- unlist(xpathApply(doc, "//code", xmlGetAttr, "codeId" )) codenames <- unlist(xpathApply(doc, "//code", xmlValue)) alldat$codes <- codenames[match(alldat$code, codeids)] # CODERS coderids <- unlist(xpathApply(doc, "//coder", xmlGetAttr, "coderId" )) codernames <- unlist(xpathApply(doc, "//coder", xmlValue)) alldat$coder <- codernames[match(alldat$coder, coderids)] # paragraph num: pgnum <- as.numeric(unlist(lapply(strsplit(paragraphCodes, "_"), function(x) x[[2]] ))) alldat$pgnum <- pgnum[match(para, paragraphIds)] # paragraph tag: paragraphTag <- unlist(xpathApply(doc, "//paragraph", xmlGetAttr, "paragraphTag")) alldat$paragraphTag <- paragraphTag[match(para, paragraphIds)]
Excellent, now we have our data in a very nice rectangular format.
Two of the most helpful diagnostics when assessing inter-coder reliability are confusion matrices and Krippendorff’s Alpha. Confusion matrices are a bit easier to produce when the data is in this format so that’s where I’ll start.
A confusion matrix is just a contingency table, or incidence matrix, that helps us figure out if any two coders are scoring things in the same way. It consists of the incidence matrix of codes for a pair of coders where the entries are the sum of the incidences—if this sounds confusing, don’t worry this will become clear in the example below. One compact way to get this is to use the paragraph-code incidence matrix for each coder, then multiply each pair of matrices. Here’s how to do it:
# get paragraph-code incidence matrix for each coder: alltabs <- table(alldat$para, alldat$codes, alldat$coder ) dimnames(alltabs)[[3]] coder1 <- alltabs[,,1] coder2 <- alltabs[,,2] coder3 <- alltabs[,,3] # Multiply together to get confusion matrix for each pair # of coders: coder12 <- t(coder1) %*% coder2 coder23 <- t(coder2) %*% coder3 coder13 <- t(coder1) %*% coder3 # Clean up column names so we can read things clearly: dimnames(coder12)[[2]] <- substr( dimnames(coder12)[[2]], 1, 6) dimnames(coder23)[[2]] <- substr( dimnames(coder23)[[2]], 1, 6) dimnames(coder13)[[2]] <- substr( dimnames(coder13)[[2]], 1, 6) # Take a look: coder12 coder23 coder13 # Pay attention to the sum on the diagonal: sum(diag(coder12)) sum(diag(coder23)) sum(diag(coder13))
Here’s what the first confusion matrix looks like:
> coder12
Adv_pa CC_exp Egregi Positi
Adv_par_const 25 0 0 6
CC_expenditure 4 12 0 6
Egregious 0 0 0 0
Position_taking_other 11 1 0 35
It shows the incidence between coder 1 and coder 2′s codes, with coder 1′s codes on the rows and coder 2′s codes on the columns. So coder 1 and coder 2 coded 25 of the same items as “Adv_par_const” but coder 1 coded “Position_taking_other” when coder 2 coded “Adv_par_const” for 11 items.
This can help diagnose which categories are creating the most confusion. We can see that our coders are confusing “Adv_par_const” and “Position_taking_other” more often than “CC_expenditure” and “Adv_par_const.” For us, that meant we focused on distinguishing these two categories in our training sessions.
It’s also useful to look at Krippendorff’s alpha to get a sense for the global agreement between all coders. We can compute Krippendorff’s alpha using the “irr” package.
But first a little data-munging is in order. The irr package expects data in a matrix with a single row for each document and columns for each coder. But of course, currently our data is in “long” format, with one line for each document-coder pair. Luckily, we can use the “reshape” package to “melt” our data then “cast” it into the format we want. In this case, “melt” does not actually change the shape of our data—it’s already long. It simply adds a “variable” column and a “value” column, which is necessary to use “cast.” Next, transform the variable to numeric so that irr will be happy. Lastly, cast the data into the format we want, with a column for each coder.
library(reshape)
alltabsm <- melt(alldat, measure.vars=c("codes"))
# Add make "value" column numeric for irr
alltabsm$value <- as.numeric(alltabsm$value)
alltabsrs <- cast(alltabsm[,which(names(alltabsm)!="code")], ... ~ coder)
And lastly, run the kripp.alpha() function on the columns that contain the coders and codes.
# KRIPP ALPHA library(irr) kripp.alpha(t(as.matrix(alltabsrs[,5:7])) )
Now, all that’s left is to get our output. What we want is to take the mode value for each article (which in this case is the same as the median). Let’s take a look at the histogram of the results.
alltabsrs$modecode <- apply(alltabsrs[,5:7], 1, median) hist(alltabsrs$modecode)
You can see from the histogram that code 3 (Eggregious) was rare, as we were expecting. The other codes look good.
And now we’ve got what we need! A data frame with each item, each code for each coder, and the most commonly occurring code that we can use as the actual label in our analysis.
Working with Bipartite/Affiliation Network Data in R [Solomon Messing] 2012-09-30 18:45

Data can often be usefully conceptualized in terms affiliations between people (or other key data entities). It might be useful analyze common group membership, common purchasing decisions, or common patterns of behavior. This post introduces bipartite/affiliation network data and provides R code to help you process and visualize this kind of data. I recently updated this for use with larger data sets, though I put it together a while back.
Much of the material here is covered in the more comprehensive “Social Network Analysis Labs in R and SoNIA,” on which I collaborated with Dan McFarland, Sean Westwood and Mike Nowak.
For a great online introduction to social network analysis see the online book Introduction to Social Network Methods by Robert Hanneman and Mark Riddle.
A network can consist of different ‘classes’ of nodes. For example, a two-mode network might consist of people (the first mode) and groups in which they are members (the second mode). Another very common example of two-mode network data consists of users on a particular website who communicate in the same forum thread.
Here’s a short example of this kind of data. Run this in R for yourself – just copy an paste into the command line or into a script and it will generate a dataframe that we can use for illustrative purposes:
df <- data.frame( person =
c('Sam','Sam','Sam','Greg','Tom','Tom','Tom','Mary','Mary'), group =
c('a','b','c','a','b','c','d','b','d'), stringsAsFactors = F)
df
person group
1 Sam a
2 Sam b
3 Sam c
4 Greg a
5 Tom b
6 Tom c
7 Tom d
8 Mary b
9 Mary d
Suppose we wish to analyze or visualize how the people are connected directly – that is, what if we want the network of people where a tie between two people is present if they are both members of the same group? We need to perform a two-mode to one-mode conversion.
To convert a two-mode incidence matrix to a one-mode adjacency matrix, one can simply multiply an incidence matrix by its transpose, which sum the common 1′s between rows. Recall that matrix multiplication entails multiplying the k-th entry of a row in the first matrix by the k-th entry of a column in the second matrix, then summing, such that the ij-th row-column entry in resulting matrix represents the dot-product of the i-th row of the first matrix and the j-th column of the second. In mathematical notation:
Notice further that multiplying a matrix by its transpose yields the following:
Because our incidence matrix consists of 0′s and 1′s, the off-diagonal entries represent the total number of common columns, which is exactly what we wanted. We’ll use the %*% operator to tell R to do exactly this. Let’s take a look at a small example using toy data of people and groups to which they belong. We’ll coerce the data to an incidence matrix, then multiply the incidence matrix by its transpose to get the number of common groups between people.
This is easy to do using the matrix algebra functions included in R. But first, you need to restructure your (edgelist) network data as an incidence matrix. An incidence will record a 1 for row-column combinations where a tie is present and 0 otherwise. One easy way to do this in R is to use the table function and then coerce the table object to a matrix object:
m <- table( df ) M <- as.matrix( m )
If you are using the network or sna packages, a network object be coerced via as.matrix(your-network); with the igraph package use get.adjacency(your-network).
This is great, but what about if we are working with a really large data set? Network data is almost always sparse—there are far more pairwise combinations of potential connections than actual observed connections. Hence, we’d actually prefer to keep the underlying data structured in edgelist format, but we’d also like access to R’s matrix algebra functionality.
We can get the best of both worlds using the Matrix library to construct a sparse triplet representation of a matrix. But we’d also like to avoid building the entire incidence matrix and just feed Matrix our edgelist directly, a point that came up in a recent conversation I had with Sean Taylor. We feed Matrix our ‘person’ column to index ‘i’ (rows in the new incidence matrix), our ‘group’ column to index j (columns in the new incidence matrix), and we repeat ’1′ for the length of the edgelist to denote an incidence.
library('Matrix')
A <- spMatrix(nrow=length(unique(df$person)),
ncol=length(unique(df$group)),
i = as.numeric(factor(df$person)),
j = as.numeric(factor(df$group)),
x = rep(1, length(as.numeric(df$person))) )
row.names(A) <- levels(factor(df$person))
colnames(A) <- levels(factor(df$group))
A
We will either convert to the ‘mode’ represented by the columns or by the rows.
To get the one-mode representation of ties between rows (people in our example), multiply the matrix by its transpose. Note that you must use the matrix-multiplication operator %*% rather than a simple astrisk. The R code is:
Arow <- A %*% t(A)
Arow will now represent the one-mode matrix formed by the row entities—people will have ties to each other if they are in the same group, in our example. Here’s what it looks like:
Arow
4 x 4 sparse Matrix of class "dgCMatrix"
Greg Mary Sam Tom
Greg 1 . 1 .
Mary . 2 1 2
Sam 1 1 3 2
Tom . 2 2 3
To get the one-mode matrix formed by the column entities (i.e. the number of people the enter the following command:
Acol <- t(A) %*% A
And the resulting co-membership matrix is as follows:
Mcol group group a b c d a 2 1 1 0 b 1 3 2 2 c 1 2 2 1 d 0 2 1 2
Although we’ve used a very small network for our example, this code is highly extensible to the analysis of larger networks with R.
Let’s work with some actual affiliation data, collected by Dan McFarland on student extracurricular affiliations. It’s a longitudinal data set, with 3 waves – 1996, 1997, 1998. It consists of students (anonymized) and the student organizations in which they are members (e.g. National Honor Society, wrestling team, cheerleading squad, etc.).
What we’ll do is to read in the data, make some mode conversions, visualize the networks in various ways, compute some centrality measures, and then compute transition probabilities (the probability that a member of one group will stay a member of the same group or become a member of a new group
# Load the "igraph" library
library("igraph")
# (1) Read in the data files, NA data objects coded as "na"
magact96 = read.delim("http://dl.dropbox.com/u/25710348/snaimages/mag_act96.txt",
na.strings = "na")
magact97 = read.delim("http://dl.dropbox.com/u/25710348/snaimages/mag_act97.txt",
na.strings = "na")
magact98 = read.delim("http://dl.dropbox.com/u/25710348/snaimages/mag_act98.txt",
na.strings = "na")
Missing data is coded as “na” in this data, which is why we gave R the command na.strings = “na”.
These files consist of four columns of individual-level attributes (ID, gender, grade, race), then a bunch of group membership dummy variables (coded “1″ for membership, “0″ for no membership). We need to set aside the first four columns (which do not change from year to year).
magattrib = magact96[,1:4] g96 <- as.matrix(magact96[,-(1:4)]); row.names(g96) = magact96$ID. g97 <- as.matrix(magact97[,-(1:4)]); row.names(g97) = magact97$ID. g98 <- as.matrix(magact98[,-(1:4)]); row.names(g98) = magact98$ID.
By using the [,-(1:4)] index, we drop those columns so that we have a square incidence matrix for each year, and then tell R to set the row names of the matrix to the student’s ID. Note that we need to keep the “.” after ID in this dataset (because it’s in the name of the variable).
Now we load these two-mode matrices into igraph:
i96 <- graph.incidence(g96, mode=c("all") )
i97 <- graph.incidence(g97, mode=c("all") )
i98 <- graph.incidence(g98, mode=c("all") )
Now, let’s plot these graphs. The igraph package has excellent plotting functionality that allows you to assign visual attributes to igraph objects before you plot. The alternative is to pass 20 or so arguments to the plot.igraph() function, which gets really messy.
Let’s assign some attributes to our graph. First we set vertex attributes, making sure to make them slightly transparent by altering the gamma, using the rgb(r,g,b,gamma) function to set the color. This makes it much easier to look at a really crowded graph, which might look like a giant hairball otherwise.
You can read up on the RGB color model here.
Each node (or “vertex”) object is accessible by calling V(g), and you can call (or create) a node attribute by using the $ operator so that you call V(g)$attribute. Here’s how to set the color attribute for a set of nodes in a graph object:
V(i96)$color[1:1295] <- rgb(1,0,0,.5) V(i96)$color[1296:1386] <- rgb(0,1,0,.5)
Notice that we index the V(g)$color object by a seemingly arbitrary value, 1295. This marks the end of the student nodes, and 1296 is the first group node. You can view which nodes are which by typing V(i96). R prints out a list of all the nodes in the graph, and those with a number are obviously different from those that consist of a group name.
Now we’ll set some other graph attributes:
V(i96)$label <- V(i96)$name V(i96)$label.color <- rgb(0,0,.2,.5) V(i96)$label.cex <- .4 V(i96)$size <- 6 V(i96)$frame.color <- NA
You can also set edge attributes. Here we’ll make the edges nearly transparent and slightly yellow because there will be so many edges in this graph:
E(i96)$color <- rgb(.5,.5,0,.2)
Now, we’ll open a pdf “device” on which to plot. This is just a connection to a pdf file. Note that the code below will take a minute or two to execute (or longer if you have a pre- Intel dual-core processor).
pdf("i96.pdf")
plot(i96, layout=layout.fruchterman.reingold)
dev.off()
Note that we’ve used the Fruchterman-Reingold force-directed layout algorithm here. Generally speaking, the when you have a ton of edges, the Kamada-Kawai layout algorithm works well but, it can get really slow for networks with a lot of nodes. Also, for larger networks, layout.fruchterman.reingold.grid is faster, but can fail to produce a plot with any meaninful pattern if you have too many isolates, as is the case here. Experiment for yourself.
Here’s what we get:

It’s oddly reminiscent of a cresent and star, but impossible to read. Now, if you open the pdf output, you’ll notice that you can zoom in on any part of the graph ad infinitum without losing any resolution. How is that possible in such a small file? It’s possible because the pdf device output consists of data based on vectors: lines, polygons, circles, elipses, etc., each specified by a mathematical formula that your pdf program renders when you view it. Regular bitmap or jpeg picture output, on the other hand, consists of a pixel-coordinate mapping of the image in question, which is why you lose resolution when you zoom in on a digital photograph or a plot produced with most other programs.
Let’s remove all of the isolates (the cresent), change a few aesthetic features, and replot. First, we’ll remove isloates, by deleting all nodes with a degree of 0, meaning that they have zero edges. Then, we’ll suppress labels for students and make their nodes smaller and more transparent. Then we’ll make the edges more narrow more transparent. Then, we’ll replot using various layout algorithms:
i96 <- delete.vertices(i96, V(i96)[ degree(i96)==0 ])
V(i96)$label[1:857] <- NA
V(i96)$color[1:857] <- rgb(1,0,0,.1)
V(i96)$size[1:857] <- 2
E(i96)$width <- .3
E(i96)$color <- rgb(.5,.5,0,.1)
pdf("i96.2.pdf")
plot(i96, layout=layout.kamada.kawai)
dev.off()
pdf("i96.3.pdf")
plot(i96, layout=layout.fruchterman.reingold.grid)
dev.off()
pdf("i96.4.pdf")
plot(i96, layout=layout.fruchterman.reingold)
dev.off()
I personally prefer the Fruchterman-Reingold layout in this case. The nice thing about this layout is that it really emphasizes centrality–the nodes that are most central are nearly always placed in the middle of the plot. Here’s what it looks like:

Very pretty, but you can’t see which groups are which at this resolution. Zoom in on the pdf output, and you can see things pretty clearly.
We’ve emphasized groups in this visualization so much, that we might want to just create a network consisting of group co-membership. First we need to create a new network object. We’ll do that the same way for this network as for our example at the top of this page:
g96e <- t(g96) %*% g96 g97e <- t(g97) %*% g97 g98e <- t(g98) %*% g98 i96e <- graph.adjacency(g96e, mode = "undirected")
Now we need to tansform the graph so that multiple edges become an attribute ( E(g)$weight ) of each unique edge:
E(i96e)$weight <- count.multiple(i96e) i96e <- simplify(i96e)
Now we’ll set the other plotting parameters as we did above:
# Set vertex attributes V(i96e)$label <- V(i96e)$name V(i96e)$label.color <- rgb(0,0,.2,.8) V(i96e)$label.cex <- .6 V(i96e)$size <- 6 V(i96e)$frame.color <- NA V(i96e)$color <- rgb(0,0,1,.5) # Set edge gamma according to edge weight egam <- (log(E(i96e)$weight)+.3)/max(log(E(i96e)$weight)+.3) E(i96e)$color <- rgb(.5,.5,0,egam)
We set edge gamma as a function of how many edges exist between two nodes, or in this case, how many students each group has in common. For illustrative purposes, let’s compare how the Kamada-Kawai and Fruchterman-Reingold algorithms render this graph:
pdf("i96e.pdf")
plot(i96e, main = "layout.kamada.kawai", layout=layout.kamada.kawai)
plot(i96e, main = "layout.fruchterman.reingold", layout=layout.fruchterman.reingold)
dev.off()
I like the Kamada-Kawai layout for this graph, because the center of the graph is too busy otherwise. And here’s what the resulting plot looks like:

You can check out the difference between each layout yourself. Here’s what the pdf output looks like. Page 1 shows the Kamada-Kawai layout and page 2 shows the Fruchterman Reingold layout.
Now we might also be interested in the percent overlap between groups. Note that this will be a directed graph, because the percent overlap will not be symmetric across groups–for example, it may be that 3/4 of Spanish NHS members are in NHS, but only 1/8 of NHS members are in the Spanish NHS. We’ll create this graph for all years in our data (though we could do it for one year only).
First we’ll need to create a percent overlap graph. We start by dividing each row by the diagonal (this is really easy in R):
ol96 <- g96e/diag(g96e) ol97 <- g97e/diag(g97e) ol98 <- g98e/diag(g98e)
Next, sum the matricies and set any NA cells (caused by dividing by zero in the step above) to zero:
magall <- ol96 + ol97 + ol98 magall[is.na(magall)] <- 0
Note that magall now consists of a percent overlap matrix, but because we’ve summed over 3 years, the maximun is now 3 instead of 1.
Let’s compute average club size, by taking the mean across each value in each diagonal:
magdiag <- apply(cbind(diag(g96e), diag(g97e), diag(g98e)), 1, mean )
Finally, we’ll generate centrality measures for magall. When we create the igraph object from our matrix, we need to set weighted=T because otherwise igraph dichotomizes edges at 1. This can distort our centrality measures because now edges represent more than binary connections–they represent the percent of membership overlap.
magallg <- graph.adjacency(magall, weighted=T) # Degree V(magallg)$degree <- degree(magallg) # Betweenness centrality V(magallg)$btwcnt <- betweenness(magallg)
Before we plot this, we should probably filter some of the edges, otherwise our graph will probably be too busy to make sense of visually. Take a look at the distribution of connection strength by plotting the density of the magall matrix:
plot(density(magall))

Nearly all of the edge weights are below 1–or in other words, the percent overlap for most clubs is less than 1/3. Let’s filter at 1, so that an edge will consists of group overlap of more than 1/3 of the group’s members in question.
magallgt1 <- magall magallgt1[magallgt1<1] <- 0 magallggt1 <- graph.adjacency(magallgt1, weighted=T) # Removes loops: magallggt1 <- simplify(magallggt1, remove.multiple=FALSE, remove.loops=TRUE)
Before we do anything else, we’ll create a custom layout based on Fruchterman.-Ringold wherein we adjust the coordates by hand using the tkplot gui tool to make sure all of the labels are visible. This is very useful if you want to create a really sharp-looking network visualization for publication.
magallggt1$layout <- layout.fruchterman.reingold(magallggt1) V(magallggt1)$label <- V(magallggt1)$name tkplot(magallggt1)
Let the plot load, then maximize the window, and select to View -> Fit to Screen so that you get maximum resolution for this large graph. Now hand-place the nodes, making sure no labels overlap:

Pay special attention to whether the labels overlap (or might overlap if the font was bigger) along the vertical. Save the layout coordinates to the graph object:
magallggt1$layout <- tkplot.getcoords(1)
We use “1″ here because only if this was the first tkplot object you called. If you called tkplot a few times, use the last plot object. You can tell which object is visible because at the top of the tkplot interface, you’ll see something like “Graph plot 1″ or in the case of my screenshot above “Graph plot 7″ (it was the seventh time I called tkplot).
# Set vertex attributes V(magallggt1)$label <- V(magallggt1)$name V(magallggt1)$label.color <- rgb(0,0,.2,.6) V(magallggt1)$size <- 6 V(magallggt1)$frame.color <- NA V(magallggt1)$color <- rgb(0,0,1,.5) # Set edge attributes E(magallggt1)$arrow.size <- .3 # Set edge gamma according to edge weight egam <- (E(magallggt1)$weight+.1)/max(E(magallggt1)$weight+.1) E(magallggt1)$color <- rgb(.5,.5,0,egam)
One thing that we can do with this graph is to set label size as a function of degree, which adds a “tag-cloud”-like element to the visualization:
V(magallggt1)$label.cex <- V(magallggt1)$degree/(max(V(magallggt1)$degree)/2)+ .3 #note, unfortunately one must play with the formula above to get the #ratio just right
Let’s plot the results:
pdf("magallggt1customlayout.pdf")
plot(magallggt1)
dev.off()
Note that we used the custom layout, which because we made part of the igraph object magallggt1, we did not need to specify in plot command.
Here’s the pdf output, and here’s what it looks like:

This visualization reveals much more information about our network than our cresent-star visualization.
Visualization series: Insight from Cleveland and Tufte on plotting numeric data by groups [Solomon Messing] 2012-03-04 13:08
After my post on making dotplots with concise code using plyr and ggplot, I got an email from my dad who practices immigration law and runs a website with a variety of immigration resources and tools. He pointed out that the post was written for folks who already know that they want to make dot plots, and who already know about bootstrapped standard errors. That’s not many people.
In an attempt to appeal to a broader audience, I’m starting a series in which I’ll outline the key principles I use when developing a visualization. In this post, I’ll articulate these principles, which combine some of Tuft’s aesthetic guidelines with Cleveland’s scientific approach to visualization, which is based on the psychological processes involved in making sense of visualizations, and has been rigorously tested via randomized controlled experiments. Based on these principles, I’ll argue that dotplots and scatterplots are better than other types of plots (especially pie charts) in most situations. In later posts, I’ll demonstrate another innovation whose widespread use I’ll credit to Cleveland and Tufte: the use of multiple panels (aka small multiples, trellis graphics, facets, generalized draftsman’s displays, multivar charts) to clearly convey the same information embedded in more complex and difficult to read visualizations, including multiple line plots and mosaic plots. In future posts I’ll also emphasize why it is important to provide some indication of the noise present in the underlying data using error bars or bands. Along the way, I’ll put you to the test–I’ll present some visualizations of the same data using different visualization techniques and ask you to try to get as much information as you can in 2 seconds from each type of visualization.
A good visualization conveys key information to those who may have trouble interpreting numbers and/or statistics, which can make your findings accessible to a wider audience (more on this below). Visualizations also give your audience a break from lexical processing, which is especially useful when you are presenting your findings–people can listen to you and process the findings from a well-designed visual at the same time, but most people have trouble listening while reading your PowerPoint bullet points. Visualizations also convey key information embedded in massive amounts of data, which can aid your own exploratory analysis of data, no matter how massive.
Yet most visualizations are flawed, drawn using elements that make it unnecessarily difficult for the human visual system to make sense of things. I see a lot of these visualizations attending research presentations, screening incoming draft manuscripts as the assistant editor for Political Communication, and as a consumer of media info-graphics (CNN is especially bad, have a look at this monstrosity). A big part of the problem is that Microsoft makes it easy to draw flashy but ultimately confusing visualizations in Excel. If you are too busy to read this post in full, follow this short list of guidelines and you’ll be on your way to producing elegant visualizations that impose a minimal cognitive burden on your audience:
The rest of the content in this series emphasizes why it makes sense to follow these guidelines. In this post I’ll look at the first point in detail and touch on the sixth. These two guidelines are most relevant when you want to look at a quantitative variable (e.g., earnings, vote-share, temperature, etc.) across different qualitative groupings (e.g., industry segment, candidate, party, racial group, season, etc.). This is one of the most common visualization tasks in business, media, and social science, and for this task people often use pie charts and/or bar charts, and occasionally dot plots.
The science of graphical perception
When most people think about visualization, they think first of Edward Tufte. Tufte emphasizes integrity to the data, showing relationships between phenomena, and above all else aesthetic minimalism. I appreciate his ruthless crusade against chart junk and pie charts (nice quote from Data without Borders). We share an affinity for multipanel plotting approaches, which he calls “small multiples,” (thanks to Rebecca Weiss for pointing this out) though I think people give Tufte too much credit for their invention—both juiceanalytics and infovis-wiki write that Cleveland introduced the concept/principle. However, both Cleveland and Tufte published books in 1983 discussing the use of multipanel displays; David Smith over at Revolutions writes that “the “small-multiples” principle of data visualization [was] pioneered by Cleveland and popularized in Tufte’s first book”; and the earliest reference to a work containing multipanel displays I could find was published *long* before Tufte’s 1983 work–Seder, Leonard (1950), “Diagnosis with Diagrams—Part I”, Industrial Quality Control (New York, New York: American Society for Quality Control) 7 (1): 11–19.
I’m less sure about Tufte’s advice to always show axes starting at zero, which can make comparison between two groups difficult, and to “show causality,” which can end up misleading your readers. Of course, the visualizations on display in the glossy pages of Tufte’s books are beautiful–they belong in a museum. But while his books are full of general advice that we should all keep in mind when creating plots, he does not put forth a theory of what works and what doesn’t when trying to visualize data.
Cleveland (with Robert McGill) develops such a theory and subjects it to rigorous scientific testing. In my last post I linked to one of Cleveland’s studies showing that dots (or bars) aligned on the same scale are indeed the best visualization to convey a series of numerical estimates. In this work, Cleveland examined how accurately our visual system can process visual elements or “perceptual units” representing underlying data. These elements include markers aligned on the same scale (e.g., dot plots, scatterplots, ordinary bar charts), the length of lines that are not aligned on the same scale (e.g., stacked bar plots), area (pie charts and mosaic plots), angles (also pie charts), shading/color, volume, curvature, and direction.
He runs two experiments: the first compares judgements about relative position (grouped bar charts) to judgements based only on length (stacked bar charts); the second compares judgements about relative position (ordinary bar charts) to judgements about angles/area (pie charts). Here are the materials he uses, courtesy of the Stanford Computer Graphics Lab:


The results are resoundingly clear—judgements about position relative to a baseline are dramatically more accurate than judgements about angles, area, or length (with no baseline). Hence, he suggests that we replace pie charts with bar charts or dot plots and that we substitute stacked bar charts for grouped bar charts.
A striking and often overlooked finding in this work is the fact that the group of participants without technical training, “mostly ordinary housewives” as Cleveland describes them, performed just as well as the group of mostly men with substantial technical training and experience. This finding provides evidence for something that I’ve long suspected: that visualizations make it easier for people lacking quantitative experience to understand your results, serving to level the playing field. If you want your findings to be broadly accessible, it’s probably better to present a visualization rather than a bunch of numbers. It also suggests that if someone is having trouble interpreting your visualizations, it’s probably your fault.
Dotplots versus pie charts and stacked barplots
Now let’s put this to the test. Take a look at each visualization below for two seconds, looking for the percent of the vote that Mitt Romney, Ron Paul, and Jon Huntsman got.
Which is easiest to read? Which conveys information most accurately? Let’s first take a look at the most critical information–the order in which the candidates placed. In all plots, the candidates are arrayed in order from highest to least vote share, and it’s easy to see that Mitt won. But once we start looking at who came in second, third, and so on, differences emerge. It’s slightly harder to process order in the pie chart because your eye has to go around the plot rather than up and down in a straight line. In the stacked bar chart, we need to look up which color corresponds to which candidate’s in the legend (as Tufte told us not to use), adding a layer of cognitive processing.
Second, which conveys estimates most accurately? The dot plot is the clear winner here. We can quickly see that Romney got about 37%, Paul got about 24%, and Huntsman got about 16%, just by looking at dots relative to the axis. When we look at the pie chart, it’s really tough to estimate the exact percent each candidate got. Same with the stacked bar chart. We could add numbers to the pie and bar charts, which would even things out to some extent, but then why not just display a table with exact percents?
One argument I used to hear all the time when I worked in industry is that pie charts “convey a sense of proportion.” Well, sure, I guess I can kind of guestimate that Ron Paul’s vote share is about 1/4. What about Jon Huntsman? Hmm, it looks like about 15 percent, which is 3/20. But wait, why do I want to convert things into fractions anyway? I don’t think in terms of fractions, I think in terms of percents. And if I really care about proportion, I suppose I could extend the axis from 0 to 100.
Suppose I want to plot results for the top 15 candidates, not just the top 6? Here’s what happens:
No contest, the pie chart fails completely. We’d need to add a legend with colors for each candidate, which adds another layer of cognitive processing–we’d need to look up each color in the lengend as we go. And even after adding the legend, you wouldn’t be able to distinguish the lower performing candidates from say write-in votes because the pie slices would be too small. The stacked bar chart will fail for the same reasons, so I’ve excluded it in the interest of brevity. Note that we don’t need to add colors to the dotplot to convey the same information, which saves an extra plotting element that we can use to represent something else (say candidate’s campaign funds or total assets). And, on top of it all, the dot plot takes up less screen/page real estate!
Why do I use dot plots instead of ordinary bar charts? A nice visualization guide from perceptualedge.com points out that often we want to only visualize differences between groups in a narrow range (they use an example wherein monthly expenses vary from $4,250-$5,500). But the length of a bar is supposed to facilitate accurate comparisons between values, so when you use a bar plot starting from $4,250, the length between bars dramatically exaggerates the actual differences. Dot plots do not have this problem because dot encode values using only location, so one must reference the axis to interpret the value.
A related points is that bars are often used to convey counts–we use them in histograms to represent frequency and track say counts of dollars earned/raised in bar charts. In fact, a team of doctors I work with at the med school recently sent in a manuscript to Radiology containing a bar chart plotting mean values between groups; they got back the following comment from the statistical reviewer: “the y-axis is quantitative but the data are represented using bars as if the data were counts.” People often use bar plots to convey estimates of means (and I’ve certainly done this), which can serve to exaggerate differences in means and hence effect sizes if you do not plot the bars from zero.
In addition, dot plots have aesthetic advantages. They convey the numerical estimate in question with a single one-dimensional point, rather than a two dimensional bar. There’s simply less that the eye needs to process. Accordingly, if a pattern across qualitative groupings exists, it’s often easier to see with a dot plot. For example, below I plot the average user ratings for each article to which Sean Westwood and I exposed subjects in a news reading experiment. The pattern that emerges is an “S” curve in which one or two stories dominate the ratings, most are sort of average, and a few are uniformly terrible. Note that you’d probably want to use something like this more for yourself than to communicate your results to others as it might overload your audience with too much information–you’d do better to select a subset of these articles or remove some of the ones in the middle (thanks to Yph Lelkes for making this point).
One question that remains is if pie charts are so bad, why are they so common? Perhaps we like them because we find them comforting just as we find pies and pizza? Well if so we’d expect pie charts to be less common in places like Japan and China where people grow up eating different food. Consider info-graphics in newspapers: I haven’t yet done a systematic content analysis, but I was unable to find a single pie chart in Japan’s Yomimuri Shimbun nor the Asahi Shimbun; nor in China’s Beijing Daily nor Sing Tao Daily. I did see plenty of maps, however, which I suppose one could argue are reminiscent of noodles.
Implementation
The most efficient way to produce solid visualizations with the ability to implement multiple panels, proper standard error estimates, and dot plots is probably in R using the ggplot2 package. If you do not have time to learn R and remain tied to MS-Excel stick to ordinary barplots to visualize quantitative variables among multiple groups (not recommended).
Otherwise, if you don’t already use it, download R and a decent editor like Rstudio. Then get started with ggplot2 and dot plots by running the following code chunk which will replicate the election figure above:
pres <- read.csv("http://www.stanford.edu/~messing/primaryres.csv", as.is=T)
# sort data in order of percent of vote:
pres <- pres[order(pres$Percentage, decreasing=T), ]
# only show top 15 candidates:
pres <- pres[1:15,]
# create a precentage variable
pres$Percentage <- pres$Percentage*100
# reorder the Candidate factor by percentage for plotting purposes:
pres$Candidate <- reorder(pres$Candidate, pres$Percentage)
# To install ggplot2, run the following line after deleting the #
#install.packages("ggplot2")
library(ggplot2)
ggplot(pres, aes(x = Percentage, y = factor(Candidate) )) +
geom_point() +
theme_bw() + opts(axis.title.x = theme_text(size = 12, vjust = .25))+
xlab("Percent of Vote") + ylab("Candidate") +
opts(title = expression("New Hampshire Primary 2012"))
After loading our data and running a few preliminary data processing operations, we pass ggplot our data set, “pres,” then we tell it what aesthetic elements we want to use, in this case that x is going to be our “Percentage” variable and y is going to be our “Candidate” variable. We tell ggplot that we want to display points for every xy pair. We also tell it to use the black and white theme, and pass some obscure axis options that ensures the axis plot correctly. Then we tell it what to label the x and y axis, and give it a title.
We can also reproduce the article ratings by story plot above using ggplot2 (even though I originally produced the plot using the lattice package).
# load the data
load(file("http://www.stanford.edu/~messing/db.Rda"))
# if you haven't installed plyr, delete the # and run this line:
# install.packages("plyr")
library(plyr)
# first we use plyr to calculate the mean rating and SE for each story
ratingdat <- ddply(db, c("story"), summarise,
M = mean(rating, na.rm=T),
SE = sd(rating, na.rm=T)/sqrt(length(na.omit(rating))),
N = length(na.omit(rating)))
# make story into an ordered factor, ordering by mean rating:
ratingdat$story <- factor(ratingdat$story)
ratingdat$story <- reorder(ratingdat$story, ratingdat$M)
# take a look at our handiwork:
# Now open a connection to a pdf file
pdf(file="plots/dotplot-story-rating.pdf", height=14, width=8.5)
ggplot(ratingdat, aes(x = M, xmin = M-SE, xmax = M+SE, y = story )) +
geom_point() + geom_segment( aes(x = M-SE, xend = M+SE,
y = story, yend=story)) +
theme_bw() + opts(axis.title.x = theme_text(size = 12, vjust = .25))+
xlab("Mean rating") + ylab("Story") +
opts(title = expression("Rating article by Story, with SE"))
dev.off()
Putting it all together: concise code to make dotplots with weighted bootstrapped standard errors [Solomon Messing] 2011-11-27 01:39
I analyze a lot of experiments and there are many times when I want to quickly look at means and standard errors for each cell (experimental condition), or the same for each cell and individual-level attribute level (e.g., Democrat, Independent, Republican). Many of these experiments are embedded in national or state-level surveys, for which each respondent is weighted so that the sample means for gender, age, and political affiliation approximate those of a probability sample. But if you use weights, it’s difficult to get unbiased estimates of the standard error of that weighted mean.
In this post, I’ll walk through how to use the powerful plyr package to summarize your weighted data, estimating weighted means and standard errors for each experimental cell using the bootstrap, and then generate dotplots to visualize the result using the ggplot2 package. Note that these are both Hadley Wickham’s packages; he’s also responsible for the immensely useful packages reshape and stringr, which I hope to write about in future posts.
First, I’ll describe the data we’re going to use. Just before the 2010 elections, my lab hired YouGov/Polimetrics to survey Illinois voters about a series of candidates for statewide office, most notably those for Secretary of State. The major party candidates included the Black Democrat incumbent, Jesse White, and a Hispanic Republican challenger, Robert Enriquez. We exposed respondents to different images of Jesse White, according to three experimental conditions: light, dark, and control (no picture). Our dependent measures included a question about how each respondent intended to vote and a feeling thermometer measuring affect toward each candidate. I use the net of the thermometer rating for Obama minus the thermometer rating for McCain (“netther”). For more on this type of measure, here’s a link to a study on feeling thermometers and their precision compared to questionnaire measures with fewer categories.
Even before we have to deal with the issue of weighted standard errors, it would be nice to find an elegant way to extract weighted statistics on subsets of our data corresponding to our experimental cells (or other qualitative grouping of our data, such as respondent party or ethnicity). It’s rather clunky and/or difficult to do this with other packages/functions, such as base::by or doBy::summaryBy because of the difficulty of passing more than one variable to the function in question, say “weighted.mean(),” applied to the subset of your data specified by a particular variable (see this discussion for details — thanks to David Freedman for the pointer to plyr::ddply). The survey::svymean function provides another potential alternative, but is nowhere near as flexible as is ddply.
We’ll use plyr::ddply, which handles this problem quite well. In the code below, we use “ddply(),” which takes a data frame for input and returns a data frame for output. The “.data” parameter specifies the data frame and the “.variables” parameter specifies the qualitative variable used to subset the data. We use “summarise()” as the function, which allows us to specify not only the function we want to pass over the subsets of our data (experimental cells), but also to tell ddply which variables in our data subset to pass to the function. For example, below we run the function “mean()” on the variable “netther” for each treatment group.
install.packages("plyr")
library("plyr")
# load the Illinois survey data
load(file("http://www.stanford.edu/~messing/data/IL2010.Rda"))
# look at ddply:
ddply(.data = IL2010, .variables= .(treat), .fun = summarise,
mean = mean(x=netther, na.rm=T),
se = sd(netther, na.rm=T)/sqrt(length(netther)) )
Now that we have some nice machinery to quickly summarize weighted data in hand, let’s get into bootstrapping. The computation of weighted standard errors for estimates of the weighted mean has no straighforward closed form solution, and hence bootstrapping is not a bad way to go. Bootstrapping consists of computing a statistic over and over, by resampling the data with replacement. For more see the UCLA stats webpage on bootstrapping in R.
I’ll use the boot package to implement bootstrapping, which requires us to specify a bootstrapping function that returns a statistic.
install.packages("boot")
library("boot")
sample.wtd.mean <- function(x, w, d) {
return(weighted.mean(x = x[d], w = w[d], na.rm=T ))
}
Why do we need this function? It allows the boot function to sample our data many times (w/ replacement), each time computing the statistic we want to estimate, as mentioned above. Specifying that function make this process quite elegant by making use of R’s indexing capabilities. Boot pass this function an index of items to include from our original data in each of the resamples. To illustrate this, run the following code in R:
playdata <- runif(20, 1, 10) playdata d <- sample(20, replace = TRUE) d playdata[d]
The code “playdata[d]” returns the “dth” element from playdata. This is exactly how the boot function works. It passes our “sample.wtd.mean()” function our index of items (d) over and over again based on sampling without replacement — utilizing the fast c code that R uses for indexing rather than slow R for-loops.
When boot is done, we take the mean or median of these statistics as our estimate, which is a loose definition of the bootstrap. We also use the standard deviation of these estimates as the estimate of the standard error of the statistic. Here’s what happens when we pass our function over our data:
b <- boot( IL2010$netther, statistic = sample.wtd.mean, R=1000, w=IL2010$weight) b sd(b$t)
[UPDATE 26 JAN 2012: perviously this was coded incorrectly. The "weights" parameter has been changed to "w." Thanks to Gaurav Sood and Yph Lelkes for pointing this out!]
Now let’s put it all together using “plyr” and “ggplot2.” I use dotplots because they convey numbers more accurately than other types of plots, such as bar plots or (worst of all) pie charts. William Cleveland has conducted research showing that dots aligned on the same scale are indeed the best visualization to convey a series of numerical estimates (Correction, the actual study is here). His definition of “best” is based on the accuracy with which people interpret various graphical elements, or as he calls them, “perceptual units.”
We’ll first just visualize results by party:
install.packages("ggplot2")
library("ggplot2")
# Create nice party ID variable:
IL2010$pid[IL2010$pid==8] <- NA
IL2010$pidcut <- cut(IL2010$pid, c(-Inf, 1, 3, 4, 6, Inf) ,
c("Strong Democrat", "Democrat", "Independent", "Republican", "Strong Republican"))
# clean up treatment variable
IL2010$treat <- factor(IL2010$treat, labels = c("Dark", "Light", "Control" ))
# Use plyr::ddply to produce estimates based on PID
pid.therm <- ddply(.data = IL2010, .variables= .(pidcut), .fun = summarise,
mean = weighted.mean(x=netther, w=weight, na.rm=T),
se = sd(boot(netther, sample.wtd.mean, R = 1000, w = weight)$t))
# plot it
ggplot(na.omit(pid.therm), aes(x = mean, xmin = mean-se, xmax = mean+se, y = factor(pidcut))) +
geom_point() + geom_segment( aes(x = mean-se, xend = mean+se,
y = factor(pidcut), yend=factor(pidcut))) +
theme_bw() + opts(axis.title.x = theme_text(size = 12, vjust = .25))+
xlab("Mean thermometer rating") + ylab("Treatment") +
opts(title = expression("Thermometer ratings for Jessie White by party"))
That’s a pretty big ggplot command, so let’s discuss each element. The first is the data to plot, which is the object produced via ddply. The second specifies the aesthetic elements of the plot, including the x and y values of the plot, and the boundaries of the plot based on specified minimum and maximum endpoints (“xmin = mean-se, xmax = mean+se”). Next, we add points to the plot by envoking “geom_point().” If we only wanted points, we could stop here. We plot lines representing standard errors with “geom_segment()” specifying x and xend. Next, we specify that we want to use theme_bw(), which I find cleaner than the default parameters. Then we make some minor adjustments via opts for aesthetics. Lastly, we set the y and x labels and the title.
Well, it’s pretty clear from our plot that there’s a lot of action on partisan ID. Let’s look at how each group is affected by the treatment. To do so, we’ll produce the plot at the top of this article, putting each partisan group in a panel via “facet_wrap(),” and plotting each treatment group. First, we’ll generate the summary data using plyr, all we need to do is add another variable (“treat”).
# PID x Treatment
trtbypid.therm <- ddply(.data = IL2010, .variables= .(treat, pidcut), .fun = summarise,
mean = weighted.mean(x=netther, w=weight, na.rm=T),
se = sd(boot(netther, sample.wtd.mean, R = 1000, w = weight)$t))
ggplot(na.omit(trtbypid.therm), aes(x = mean, xmin = mean-se, xmax = mean+se, y = factor(treat))) +
geom_point() + geom_segment( aes(x = mean-se, xend = mean+se,
y = factor(treat), yend=factor(treat))) +
facet_wrap(~pidcut, ncol=1) +
theme_bw() + opts(axis.title.x = theme_text(size = 12, vjust = .25))+
xlab("Mean thermometer rating") + ylab("Treatment") +
opts(title = expression("Thermometer ratings for Jessie White by treatment condition and pid"))
It looks like Republicans generally don’t like the dark image, while Democrats do. But what if we want to look at the way that racial attitudes interact with the treatments? We measured implicit racial attitudes via the IAT). Let’s take a look.
# Fix up the data to remove the "Control" condition and all "na's"
ilplot <- IL2010[-which(is.na(IL2010$pidcut)),]
ilplot$treat[ilplot$treat=="Control"] <- NA
ilplot$treat <- ilplot$treat[drop=T]
ilplot <- ilplot[-which(is.na(ilplot$treat)),]
ilplot$dscore[which(ilplot$dscore< -1)] <- NA
# Plot it
ggplot(ilplot, aes(x = dscore, y = netther, colour=treat)) +
geom_point() + geom_smooth(method = "loess", size = 1.5) +
facet_wrap(~pidcut, nrow=1) +
theme_bw() + opts(axis.title.x = theme_text(size = 12, vjust = .25))+
xlab("IAT d-score (- pro black, + anti-black)") + ylab("Thermometer rating") +
opts(title = expression("Thermometer ratings for Jessie White by treatment condition and iat"))
With this plot, we don’t really need to subset the data because we are using all of the points and just using lowess to draw lines representing the conditional means across every value of IAT score. The plot shows what seems to be an interaction between IAT score and treatment, such that those with high (anti-black) IAT scores generally favor the lighter skinned image.
Map the distribution of your sample by geolocating ip addresses or zip codes [Solomon Messing] 2011-09-18 17:53
At any rate, we made a map that looks something like the picture above.
In this post I’ll illustrate how to make a map like this first using zip+4 codes and then using ip addresses. We’ll use RCurl to send an html POST command to some website that will give us the relevant geolocation data, and then extract the relevant info by parsing JSON or html output (using regular expressions).
First we’ll pass zip+4 codes to the Yahoo! PlaceFinder Application Programming Interface (API) to return the state, latitude and longitude. We’ll use their JSON output, because it’s easier to parse in R than the alternative (XML).
The first thing you’ll need to do is to get yourself a Yahoo! Application ID, so that you can use Yahoo’s API. This should only take about ten minutes. When you’ve got it, replace your appid where it says “[INSERT YOUR YAHOO ID HERE]” in the code below.
Here’s the R code:
###############################################################################
################## Example 1: Geolocating Zip +4 Codes ########################
###############################################################################
zips <- c("48198-6275", "75248-5000", "27604-1520", "53010-3212", "95453-9630",
"10548-1241", "70582-6107", "84106-2494", "78613-3249", "18705-3906",
"23108-0274", "23108-0274", "67361-1732", "28227-1454", "37027-5902",
"75067-4208", "32505-2315", "80905-4449", "49008-1818", "08034-2616",
"04347-1204", "94703-1708", "32822-4969", "44035-6205", "60192-1114",
"45030-4933", "32210-3625", "80439-8782", "68770-7013", "42420-8832",
"83843-2219", "33064-2555", "75067-6605", "22301-1266", "78757-1652",
"85140-5232", "19067-6225", "06410-4210", "63109-3347", "89103-4803",
"99337-5714", "37067-8123", "94582-4921", "65401-3065", "46614-1320",
"29732-3810", "79412-1142", "23226-3006", "78217-1542", "10128-4923",
"15145-1635", "48135-1093", "50621-0683", "32713-3803", "08251-1332",
"14445-2024", "83210-0054", "27525-8790", "32609-8860", "53711-3548",
"49601-2214", "15324-0010", "37604-3508", "85041-7739", "01906-1317",
"10552-2605", "20120-6322", "19934-1263", "87124-2735", "22192-4415",
"45344-9130", "08618-1464", "83646-3473", "80911-9007", "13057-2023",
"30033-2016", "30039-6323", "20109-8120", "98043-2105", "34655-3129",
"60465-1326", "33433-5746", "30707-1636", "98102-4432", "92037-7407",
"95054-4144", "94703-1708", "94110-2712", "92562-5073", "31418-0341",
"10009-1819", "73703-6736", "98554-0175", "65649-8267", "21704-7864",
"15209-2420", "61550-2715", "92506-5317", "60611-3801", "48161-4064",
"77365-3268", "50022-1388", "63841-1227", "36207-5817", "22121-0232",
"73115-4446", "85340-7341", "44420-3415", "07663-4913", "10065-6716",
"91606-2213", "19120-9237", "80015-2718", "97401-4448", "46637-5424",
"29609-5425", "20815-6414", "45373-1731", "57106-6205", "71744-9486",
"14222-1922", "28306-3901", "84074-2684", "28605-8292", "59405-8649")
###############################################################################
############################# Geolocate data ##################################
###############################################################################
# First load RCurl, which we'll use to send HTML POST commands to the Yahoo! API
library(RCurl)
# Next load RJSONIO, an R library that deals with JavaScript Object Notation (JSON)
# Input/Output. In my experience this library is a bit faster and more robust than RJSON.
library(RJSONIO)
# create variables to store output:
state <- character(length(zips))
lat <- character(length(zips))
lon <- character(length(zips))
appid <- "&appid=[INSERT YOUR YAHOO ID HERE]"
# try a sample query
ipinfo <- fromJSON(getURL(paste("http://where.yahooapis.com/geocode?flags=J&country=USA&q=", zips[1], appid, sep="")))
# the good stuff is here:
ipinfo$ResultSet$Results[[1]]
for(i in 1:length(zips)){
#i=1
print(paste("i = ", i, " zip = ", zips[i] ))
# Read in geolocation data
ipinfo <- fromJSON(getURL(paste("http://where.yahooapis.com/geocode?flags=J&country=USA&q=", zips[i], appid, sep="")))
if(as.numeric(ipinfo$ResultSet[6])>0){
# state
try(suppressWarnings(state[i] <- ipinfo$ResultSet$Results[[1]][24]))
print(ipinfo$ResultSet$Results[[1]][24])
# get lattitude
try(suppressWarnings(lat[i] <- ipinfo$ResultSet$Results[[1]][2]))
print(ipinfo$ResultSet$Results[[1]][2])
# get longitude
try(suppressWarnings(lon[i] <- ipinfo$ResultSet$Results[[1]][3]))
print(ipinfo$ResultSet$Results[[1]][3])
}
}
###############################################################################
####################### draw maps based on ip addresses #######################
###############################################################################
# We'll draw points for each observation of lattitude and longitude, and
# we'll shade in each state according to how many cases we have in each state
# first count the number of cases in each state:
mtstates <- table(state)
mtstates
# Convert state abbreviations to full state names:
mtfullstatenames <- tolower(state.name[match(names(mtstates), state.abb)])
mtfullstatenames
mtfullstatenames[names(mtstates)=="DC"] <- "district of columbia"
names(mtstates) <- mtfullstatenames
# use "cut" to group the count data into several groups
mtstatesb <- cut(mtstates, breaks= c(-Inf, 0, 1, 4, 7, Inf), labels=c("0", "1", "2-4", "5-7", "8+") )
mtstatesb
library(maps)
# generate a map object so that we can match names to the state level shape files
usmap <- map("state")
# Some of the state level shape files are broken down by sub-state region. We
# just care about the political boundaries for the purposes of this project,
# so we will not differentiate. Thus, we need to make all names within a
# state the same, which will mean some names are repeated.
# Luckily the usmap$names are as follows: state:subregion
usmap$names
# so we can just split the strings based on the ":" character. We'll use
# sapply( ..., "[[", 1) which extracts the first element when a list is returned:
mapstates <- sapply(strsplit(usmap$names, ":"), "[[", 1)
mapstates
# Now we need to generate a color for each item in mapstates so we can color in the map.
# We first use "match()" to index our count of observations per state in mtstatesb by mapstates.
# shapeFileN will contain the numeric range specified in mstatesb, but indexed by the
# the names in mapstates, some of which will of course be repeated as discussed above.
shapeFileN <- mtstatesb[match(mapstates,names(mtstates))]
# Now we've can generate a series of grey colors indexed by the count of observations per state,
# indexed so that the the observations match the shape files that come with the "state" object in
# the maps package:
cols <- rev(grey.colors(5))[shapeFileN]
# now let's draw the map:
png("demosamplemap.png", width=1000, height=700)
# draw the map object using the colors specified above
map("state", col= cols, fill=T)
# draw lattitude and longitude points for each observation:
points(lon, lat, cex = .3, col = rgb(0,0,0,.5))
# draw a legend
legend("bottomright", levels(mtstatesb), fill = c("white", rev(grey.colors(5))[2:5]), bty="n")
# put a title on it:
title("Geographic distribution of Zip+4 codes")
dev.off()
Note that for publication purposes, you’ll want to create a pdf instead of a png file.
OK, now for our second example, which will geolocate using ip addresses. I found an excellent ip database website, it’s pretty current and has nicely formatted html: http://www.ip-db.com/. You can lookup an ip address as follows: http://www.ip-db.com/%5Bipaddress%5D. Take a look at the html for http://www.ip-db.com/64.90.182.55, which is one of the NIST Internet Time Servers, which I’ll be using to create example code. If you view the html source, you’ll see that the relevant state is embedded in this line:
Region: <span style="font-family: arial; font-size: x-small;">New York
Also embedded is the lattitude and longitude, which we will need to create the map:
Lat/Long: <span style="font-family: arial; font-size: x-small;"><a href="http://maps.google.com/maps?om=1&q=40.7089+-74.0012&z=10" target="_blank">40.7089 -74.0012</a>
Now we’ll just use RCurl to send a set of ip addresses to this website, then use Regular Expressions to extract the relevant data from the html that the website returns. Code below:
###############################################################################
################## Example 2: Geolocating IP Addresses ########################
###############################################################################
ips <- c("64.90.182.55", "96.47.67.105", "206.246.122.250", "129.6.15.28", "64.236.96.53",
"216.119.63.113", "64.250.177.145", "208.66.175.36", "173.14.55.9", "64.113.32.5",
"66.219.116.140", "24.56.178.140", "132.163.4.101", "132.163.4.102", "132.163.4.103",
"192.43.244.18", "128.138.140.44", "128.138.188.172", "198.60.73.8", "64.250.229.100",
"131.107.13.100", "207.200.81.113", "69.25.96.13", "216.171.124.36", "64.147.116.229")
# create variables to store output:
state <- character(length(ips))
lat <- character(length(ips))
lon <- character(length(ips))
# iterate over ip addresses:
for(i in 18:length(ips)){
#i=5
print(paste("i = ", i, " ip: ", i))
ipinfo <- getURL(paste("http://www.ip-db.com/", ips[i], sep=""))
# use RegEx to extract key info:
library(stringr)
# Get state
# We use the regex (\\w\\s?)+ where \\w = word character, \\s? = possible space character,
# () groups the pattern, and + means one or more.
# note that we must escape " characters by \" to include them in the pattern
pattrn <- "Region:</b></font></td><td width=\"10\"> </td><td><font face=\"arial\" size=\"2\">(\\w\\s?)+</td>"
( results <- str_extract(ipinfo, pattrn))
# Extract the actual text of the state and clean it up so we can actually use it:
state[i] <- str_extract(results, ">(\\w\\s?)+<")
state[i] <- gsub(">", "", state[i])
state[i] <- gsub("<", "", state[i])
print(state[i])
# Now get lattitude and longitude:
# We can limit our pattern to target="_blank" because it's a unique pattern in the html
# An example of our substring of interest follows: "target=\"_blank\">40.7089 -74.0012</a></td>"
# So we use this pattern \\-?\\d\\d(\\.\\d+)? \\-?\\d\\d(\\.\\d+)? where \\d means digit,
# means optional, () groups the pattern, and \\- is an escaped dash
pattrn <- "target=\"_blank\">\\-?\\d+(\\.\\d+)? \\-?\\d+(\\.\\d+)?</a></td>"
( results <- str_extract(ipinfo, pattrn))
# Extract both lattitude an longitude and clean it up so we can save it as numeric in the data:
latlon <- str_extract_all(results, "\\-?\\d+(\\.\\d+)?")
# the code above returns a list in case results has more than one set of characters in the character
# vector, so we need to unlist it to make it into a normal vector:
latlon <- unlist(latlon)
# since there are two results, we return one for each for lat and lon:
lat[i] <- latlon[1]
lon[i] <- latlon[2]
print(lat[i])
print(lon[i])
# Put a delay between entries so there's no danger of overwhelming the server
Sys.sleep(sample(15:25,1)/10)
}
###############################################################################
####################### draw maps based on ip addresses #######################
###############################################################################
# We'll draw points for each observation of lattitude and longitude, and
# we'll shade in each state according to how many cases we have in each state
# first count the number of cases in each state:
mtstates <- table(state)
# then match to the state.name object (which the maps object also uses)
mtfullstatenames <- tolower(state.name[match(names(mtstates), state.name)])
# extract the state names to a separate object:
names(mtstates) <- mtfullstatenames
# use "cut" to group the count data into several groups
mtstatesb <- cut(mtstates, breaks= c(-Inf, 0, 1, 2, 4, Inf), labels=c("0", "1", "2", "3-4", "4+") )
library(maps)
# generate a map object so that we can match names to the state level shape files
usmap <- map("state")
# Some of the state level shape files are broken down by sub-state region. We
# just care about the political boundaries for the purposes of this project,
# so we will not differentiate. Thus, we need to make all names within a
# state the same, which will mean some names are repeated.
# Luckily the usmap$names are as follows: state:subregion
usmap$names
# so we can just split the strings based on the ":" character. We'll use
# sapply( ..., "[[", 1) which extracts the first element when a list is returned:
mapstates <- sapply(strsplit(usmap$names, ":"), "[[", 1)
mapstates
# Now we need to generate a color for each item in mapstates so we can color in the map.
# We first use "match()" to index our count of observations per state in mtstatesb by mapstates.
# shapeFileN will contain the numeric range specified in mstatesb, but indexed by the
# the names in mapstates, some of which will of course be repeated as discussed above.
shapeFileN <- mtstatesb[match(mapstates,names(mtstates))]
# Now we've can generate a series of grey colors indexed by the count of observations per state,
# indexed so that the the observations match the shape files that come with the "state" object in
# the maps package:
cols <- rev(grey.colors(5))[shapeFileN]
# now let's draw the map:
png("demosamplemap.png", width=1000, height=700)
# draw the map object using the colors specified above
map("state", col= cols, fill=T)
# draw lattitude and longitude points for each observation:
points(lon, lat, cex = .3, col = rgb(0,0,0,.5))
# draw a legend
legend("bottomright", levels(mtstatesb), fill = c("white", rev(grey.colors(5))[2:5]), bty="n")
# put a title on it:
title("Geographic distribution of NIST Internet Time Servers")
dev.off()
Trinity, Oases, SOAPdenovo-trans – Many Helpful Tips from Richard Smith [Homologus] 2013-10-31 12:26
Reposting here -
The post Trinity, Oases, SOAPdenovo-trans – Many Helpful Tips from Richard Smith appeared first on Homologus.
A Video Dedicated to #ENCODE, #GWAS and Other Government-funded ‘Big Science’ Projects [Homologus] 2013-10-31 00:37
Michael Schatz mentioned that ENCODE leader Mike Snyder will be presenting at the #GI2013 meeting, but his name is nowhere in the list of presenters.
Have no worry !! Through our NSA and other spying channels, we received a video describing his research.
For those looking for less serious content than the above video, the following map shows the locations all next-gen sequencing machines around the world (click on the image to see google map).
For some strange reason, none is listed in Russia. Did they all go offline since Snowden’s arrival?
The post A Video Dedicated to #ENCODE, #GWAS and Other Government-funded ‘Big Science’ Projects appeared first on Homologus.
Genome Informatics Meeting at CSHL #GI2013 [Homologus] 2013-10-30 21:58
The meeting is taking place currently. Please follow #GI2013 at twitter for running commentary. A list of talks and posters are given below from this page.
|
Presenting Author |
Abstract Title |
Talk/Poster |
Abolude, O. |
Human Microbiome Project data |
poster |
Adhikari, B. |
Genomic analysis of atoxigenic |
poster |
Aganezov, S. |
Varying-resolution synteny blocks |
poster |
Ainsley, J.A. |
Genome wide characterization of |
talk |
Aitken, S. |
Kinetic signatures—A novel |
poster |
Akatsuka, S. |
Informatics for analyzing |
poster |
Alameer, R.S. |
Modeling complex genetic and |
poster |
Alekseyev, M. |
Reconstruction of ancestral |
talk |
Argimon, S. |
In silico genome subtraction |
poster |
Ballouz, S. |
Characterizing RNA-seq through the |
poster |
Barrell, D. |
Ensembl Gene Annotation 2013 |
poster |
Beier, S. |
Assembling barley chromosome 3H by |
poster |
Benoukraf, T. |
Visualizing the DNA/DNA |
poster |
Berthelot, C. |
Benchmarking ChIP-seq pipelines |
poster |
Blankenberg, D. |
Wrangling Galaxy’s reference data |
poster |
Bouvier, E. |
Improving reproducibility using |
poster |
Boyle, A.P. |
The real effect of SNPs on |
poster |
Brody, T. |
Tools for analysis and comparison |
talk |
Canzar, S. |
Cutterhead—Recovering |
poster |
Carmel, L. |
Paleo-epigenetics—Reconstructing |
talk |
Chan, P.P. |
RNA-seq analysis workflow |
poster |
Chin, J. |
String graph assembly for diploid |
talk |
Coffman, A. |
DGIdb—Mining the druggable genome |
poster |
Colak, R. |
ELASPIC—Combining ensemble |
talk |
Colak, R. |
Novel machine learning approach |
poster |
Coraor, N. |
Galaxy’s long-term |
talk |
Criscione, S.W. |
RepEnrich—A new method to estimate |
poster |
Daugherty, S.C. |
The IGS analysis engine |
poster |
Davila, J. |
Analysis and characterization of |
poster |
Day, D.S. |
Responsive histone modifications |
talk |
De Pons, J.L. |
Search, visualization, comparison, |
poster |
DeBoever, C.M. |
Transcriptome sequencing reveals |
poster |
Denas, O. |
Deep modeling of gene expression |
poster |
Dobin, A. |
Circular RNA detection and |
poster |
Dror, I. |
Widespread evidence for DNA shape |
talk |
Erickson, D.T. |
ENCODE Project data access via |
poster |
Erlich, Y. |
Genetic media |
talk |
Ferretti, V. |
The new ICGC data portal and its |
poster |
Fiume, M. |
MedSavant—Graphical search engine |
talk |
Fortini, E. |
Optimization of PAR-CLIP and |
poster |
Fourrage, C. |
Identification and visualization |
poster |
Frankish, A. |
Identifying functional ‘nonsense’ |
poster |
Friedberg, I. |
Critical assessment of function |
poster |
Fu, F. |
RNA-Seq based transcriptome |
poster |
Fu, Y. |
Conserved secondary structure |
poster |
Garber, M. |
Evolutionary dynamics and tissue |
talk |
Gerstein, M.B. |
Integrative annotation of variants |
talk |
Ghosh, S. |
Evaluation of methods to analyze |
poster |
Ghosh, S. |
Two-step alignment for optimal ION |
poster |
Giardine, B.M. |
Using workflows for consistent |
poster |
Goecks, J. |
Understanding cancer genomes using |
poster |
Gonnella, G. |
Harlekin—Effective and scalable |
poster |
Gonzalez, J.M. |
Increasing the GENCODE mouse |
poster |
Gout, A. |
Transcriptome analysis reveals |
poster |
Gupta, V. |
GABox—A “white-box” genome |
poster |
Gurtowski, J. |
An improved method for hybrid |
poster |
Gymrek, M.A. |
Short tandem repeat polymorphisms |
talk |
Haberman Ziv, Y. |
Ileal transcriptome analysis in |
poster |
hajirasouliha, i. |
A combinatorial approach for |
poster |
Halpern, A. |
Use of known variants within the |
poster |
Hansen, N.F. |
BARD—Detection of copy number |
poster |
Hara, Y. |
Identification and |
talk |
Hayden, K.E. |
Complete sequence representation |
talk |
Hayes, R.D. |
Transitioning phytozome genome |
poster |
Herrero, J. |
Ancestry of single base pair |
talk |
Herrero, J. |
Genome assembly assessment based |
poster |
Hide, W. |
Community development of validated |
talk |
Hon, C. |
Determinants for the localization |
talk |
Hong, S. |
Draft genome sequence of three |
poster |
Hou, M. |
Detecting pico-inversions using |
poster |
Imai, T. |
New genome assembly algorithm |
poster |
Isomoto, A. |
Population based discovery of |
poster |
Jacobsen, A. |
Systematic analysis of microRNA |
poster |
Jain, D.P. |
SBRI—A sampling based quantitative |
poster |
Janga, S. |
Dissecting the expression |
poster |
Janin, L. |
Versatile cloud-based genomics |
poster |
Janky, R. |
Detecting master regulators and |
poster |
Jex, A.R. |
The draft Trichuris suis |
poster |
Jiao, Y. |
Maize Pan-genome construction by |
poster |
Jose, A. |
Functional annotation of |
poster |
JUNG, B. |
Bacterial community structure and |
poster |
Kannan, S. |
Optimal algorithms and fundamental |
talk |
Kasowski, M. |
Extensive variation in chromatin |
talk |
Katayama, K. |
Histogram Clustering approach for |
poster |
Khalfan, M. |
DNA subway—Genomics, DNA |
poster |
Kim, D. |
TopHat3—Faster and more sensitive |
poster |
Kucukural, A. |
Flexible pipeline generation |
poster |
Kumari, P. |
Emergence—Data-driven pipeline |
poster |
Kyriazopoulou Panagiotopoulou, S. |
Integrating gene expression and |
poster |
Lam, E. |
Mapping of a non-canonical |
poster |
Lau, B. |
Discovering more variants in a |
talk |
Layer, R.M. |
Efficient and accurate DNA |
poster |
Layer, R.M. |
LUMPY—A probabilistic framework |
poster |
Lederman, R. |
General purpose and customized |
poster |
Lederman, R. |
Using the long-range |
poster |
Li, J. |
Oshell—A comprehensive work |
poster |
Li, Q. |
A regression model for assessing |
poster |
Liseron-Monfils, C.V. |
The dynamic of regulatory network |
poster |
Lister, R. |
Global epigenomic reconfiguration |
talk |
Liu, X. |
Linear time de novo detection of |
poster |
Loraine, A. |
Visualizing RNA-Seq data with |
poster |
Ma, J. |
Tracing the evolution of |
talk |
MacArthur, D.G. |
Integrated analysis of |
talk |
Marques, A. |
Chromatin status separates two |
talk |
MARSHALL, J. |
The future of the Samtools |
poster |
Marshall, M. |
Exome sequencing 1000 individuals |
poster |
Martin, F. |
Collared flycatcher genome |
poster |
Mascher, M. |
Anchoring and ordering NGS contig |
talk |
Middleton, S.A. |
NoFold—RNA structure |
poster |
Miller, J.R. |
Scalable assembly of native |
talk |
| Minot, S. | Evaluating novel metagenomic classification algorithms for forensic microbial detection |
poster |
| Mirmomeni, M. | Increasing genome assembly quality using high performance computing |
poster |
Monaco, M.K. |
Gramene—A resource for comparative |
poster |
Moore, B.L. |
The antecedents of higher-order |
poster |
Morrissey, C.S. |
Improving ChIP-seq peak |
poster |
Mortazavi, A. |
A computational pipeline for the |
talk |
Moxon, S. |
A combined computational and |
poster |
Mukamel, E.A. |
Genomic sequence determinants of |
poster |
Naito, Y. |
GGRNA—A Google-like, ultrafast |
poster |
Nakaki, R. |
CoLo—A novel algorithm to |
poster |
Nakato, R. |
Comparative ChIP-seq analysis of |
poster |
Narechania, A. |
Finding clusters in the flock—A |
poster |
Narzisi, G. |
SCALPEL—Micro-assembly approach to |
talk |
Nekrutenko, A. |
Finding missing analysis tools |
poster |
Neretti, N. |
Genome-wide transcriptional |
talk |
Ning, Z. |
Cross_genome—Assembly Improvement |
poster |
Nip, K. |
jhive—Visual network comparison |
talk |
Nissen, A.M. |
Trio-based study of neuromuscular |
poster |
Niu, B. |
HOTSPOT—A novel computational tool |
poster |
Noutsos, C. |
Atmosphere, iPlant Collaborative’s |
poster |
Nutter, N.G. |
Progress towards a turnkey system |
talk |
O’Connor, B.D. |
SeqWare on the multicloud—Enabling |
poster |
Oesper, L. |
Analysis of complex genomic |
talk |
Olson, A. |
Learning to tell the difference |
poster |
Ono, H. |
RefEx—Reference expression dataset |
poster |
Onuki, R. |
Development of a miRNAs prediction |
poster |
O’Rawe, J.A. |
Integrating multiple sequencing |
poster |
Ouellette, F. |
Data availability and re-usability |
talk |
Ouellette, F. |
FGED—The Functional Genomics Data |
poster |
Ouellette, F. |
Streamlining development and |
poster |
Park, G. |
De novo assembly and |
poster |
Patro, R. |
Sailfish—RNA-seq expression |
talk |
Pendleton, M.R. |
Detection and resolution of tandem |
poster |
Philip, G. |
Bioinformatics tutorials using |
poster |
Piper, J. |
Wellington—A novel method for the |
poster |
Prasad, A. |
Genome sequence and initial |
poster |
Preece, J.S. |
Plant Reactome—Metabolic and |
poster |
Price, T.S. |
Benchmarking RNAseq algorithms to |
poster |
Quinlan, A.R. |
Mining genetic variation with |
talk |
Raney, B.J. |
Assembly data hubs support viewing |
poster |
Rao, K.R. |
Functional data-mining for protein |
poster |
Ratsch, G. |
Causes and consequences of cancer |
talk |
Ratsch, G. |
PALMapper—Fast, accurate and |
poster |
Ream, D. |
Towards a universal model of gene |
poster |
Rebolledo-Jaramillo, B. |
Controlling for contamination in |
poster |
Regier, A. |
Automatic interpretation of |
poster |
Rensch, T. |
Modelling of complex tissue |
poster |
Rockowitz, S. |
Comparison of REST cistromes |
poster |
Roest Crollius, H. |
Ancestral reconstructions provide |
talk |
Ronen, R. |
Whole genome sequencing uncovers |
talk |
Rozowsky, J. |
Comparison of 3 metazoan |
talk |
Saha, S. |
Composition of the maize |
poster |
Sanchez-Vega, F. |
Differential methylation in the |
poster |
Sankoff, D. |
Genome aliquoting for ancient |
poster |
Sauria, M. |
High-resolution HiC analysis |
poster |
Sedlazeck, F.J. |
Improving de novo genome and |
poster |
Sese, J. |
Statistical significance of |
poster |
Shah, R.H. |
Developing a framework for for |
poster |
Shahid, S. |
De novo annotation of |
poster |
Shimamura, T. |
Hierarchical Bayes model-based |
poster |
Shu, S. |
PERTRAN—Predict transcripts from |
poster |
Sibbesen, J.A. |
Probabilistic transcriptome |
talk |
Silverstein, K.A. |
Detecting small plant peptides |
poster |
Singer, M. |
Intragenic exon methylation |
poster |
Smith, S. |
The Genome Modeling System—An |
talk |
Sompallae, R. |
Clinical validation of targeted |
poster |
Song, G. |
Comparative analysis of |
poster |
Song, L. |
CLASS—A program for accurate |
poster |
Stadler, M. |
Systematic exploration of coding |
poster |
Standage, D.S. |
mRNAmarkup—Quality control and |
poster |
Stanke, M. |
Simultaneous gene finding in |
talk |
Syed, A. |
Developing a data management |
poster |
Taghavi, Z. |
Distilled single-cell genome |
poster |
Tang, L. |
Visualizing consequences of |
poster |
Thiru, P. |
Mining expression compendia to |
poster |
Trinh, J. |
Genome-wide linkage analysis and |
poster |
Verleyen, W. |
Characterizing algorithmic and |
poster |
Wang, H. |
Novel stress-induced smRNAs from |
poster |
Wang, Q. |
The core regulons orchestrating |
poster |
Wei, S. |
Exploring rice phylogenomics with |
poster |
Westholm, J.M. |
Identification of thousands of |
poster |
Whitley, P. |
Effects of sequencing coverage |
poster |
Wong, E. |
A metadata standard to improve |
poster |
Wong, E. |
Allele-specific contributions to |
poster |
Wong, W. |
Whole genome miRNA-mRNA-DNA |
poster |
Wood, D.E. |
Rapid phylogenetic sequence |
talk |
Wu, T. |
Hybrid strategies for aligning |
poster |
Xuan, Z. |
Reveal significant association of |
poster |
Yan, K. |
OrthoClust—An orthology-based |
talk |
Yeung, J. |
Whole transcriptome alternative |
poster |
ZaÏd, N. |
Coverage rate of ADME genes from |
poster |
Zerbino, D.R. |
Updating the Ensembl regulatory |
poster |
Zhang, J. |
Classify breast cancer patients |
poster |
Zhang, W. |
Competition between Pol II and |
poster |
Zhao, S. |
TMM_RPKM for within- and |
poster |
Zhao, X. |
A hybrid method of incorporating |
poster |
Zhu, Y. |
A framework to generate ortholog |
poster |
Zhu, Y. |
Comprehensive depiction of genomic |
poster |
Zmasek, C.M. |
Analysis of the evolution of the |
poster |
The post Genome Informatics Meeting at CSHL #GI2013 appeared first on Homologus.
A Few Useful Terms [Homologus] 2013-10-30 19:25
Publishing
Getting your research report locked up so that the public cannot see.
Publishing in high visibility journal
Getting your research report removed from public view for ever.
Recently we tried to access a 1951 paper at Nature and, to our utter surprise, found the journal keeping it locked up 62 years after ‘publication’ !!! Copyright laws, initially created to protect individual authors, now allow these publishing houses to do whatever they want.
Science Paper
Fictional text, which the authors pay to get published (‘publish’ defined as above).
Scientist
A derogatory term for PI (principal investigator). For example, you can insult someone by saying “he is only a scientist, not a PI”. Fifteen years ago, the opposite statement could have been an insult.
Higher Organism
An organism with higher level of government funding.
Lower Vertebrate
A vertebrate NIH and other funding agencies does not care about.
Bioinformatician
A technician or scientist (used in derogatory way), who can run computer programs to analyze ‘big science’ data.
Bioinformatics Guru
A technician or scientist (used in derogatory way), who can run the above programs from command line.
Bioinformatics PI
So rare that he is given a unique name – Titus Brown. Only person with more extraordinary abilities than him is Kanye West.
Personal Genomics
Eugenics for the ipad generation.
New Study Reveals
New study failed utterly except in its ability to fool a journalist. For a detailed list of revealing studies, check
“Shocking Finding that a Genome by Itself Provides Little Insight”
College Student
A kid so weak in math that he cannot comprehend the compounding formula in his student loan contract.
Graduate Student
When the above kid finally reads his student loan contract.
Did we miss something?
The post A Few Useful Terms appeared first on Homologus.
A Cuban Blog on Bioinformatics and Proteomics [Homologus] 2013-10-29 03:20

Yasset Perez-Riverol, who works as a bioinformatician in Cuba, contacted us by email to mention his informative blog that our readers may enjoy. Below we provide links to couple of his commentaries. Please feel free to give feedback to him here or in his blog.
Celebrating Ten Years of Mann and Aebersold’s “Mass spectrometry-based proteomics” review
In 2003 Mann & Aebersold reviewed on the pages of Nature the challenges and perspectives of the then-nascent field of MS-based proteomics. Mass spectrometry (MS) has since entrenched itself as the method of choice for analyzing complex protein samples, and MS-based proteomics has become an indispensable technology for interpreting genomic data and performing protein analyses (primary sequence, post-translational modifications (PTMs) or protein–protein interactions).
“The ability of mass spectrometry to identify and, increasingly, to precisely quantify thousands of proteins from complex samples can be expected to impact broadly on biology and medicine.”
The manuscript by Mann & Aebersold is one of the most cited manuscripts in the field of MS proteomics, For this reason is one of the “core papers” in the field of proteomics and computational proteomics, outlining most of the basic concepts required to understand the fundamentals of this discipline.
Why R for Mass Spectrometrist and Computational Proteomics
Why R:
Actually, It is a common practice the integration of the statistical analysis of the resulted data and in silico predictions of the data generated in your manuscript and your daily research. Mass spectrometrist, biologist and bioinformaticians commonly use programs like excel, calc or other office tools to generate their charts and statistical analysis. In recent years many computational biologists especially those from the Genomics field, regard R and Bioconductor as fundamental tools for their research.
R is a modern, functional programming language that allows for rapid development of ideas; it is a language and environment for statistical computing and graphics.The rich set of inbuilt functions makes it ideal for high-volume analysis or statistical studies.
We find it quite fascinating that scientific researchers from around the world are using the internet to get connected to each other and share ideas without going through the journals acting as intermediaries. That means there will be a continuum in the modes of communications and all scales, ranging from short tweets all the way up to journal publications in highly polished languages, will be seen as viable means of communication.
The post A Cuban Blog on Bioinformatics and Proteomics appeared first on Homologus.
Fractals and Scale-free Networks [Homologus] 2013-10-29 02:20
In science, what we see is limited by what our mathematics allows us to see. The mathematical framework built since Newton’s time has been linear and our observations and ways of analysis are restricted by that linear framework. Around the middle of last century, Mandelbrot and others did extensive work to understand nonlinear contributions in dynamical systems and showed that nonlinearity gave rise to fractals and chaos.
A number of papers were published by Barabasi’s lab around 1999-2000. They showed that protein-protein interaction networks were ‘scale-free‘, which is a strong signature of nonlinearity. Those observations should have profound implication in the analysis of biological systems, but the biologists responded with ‘so what’.
Barabasi’s papers inspired me to start working in biology, and in our first two papers, we tried to answer the ‘so what’ question. We showed that the scale-free networks were not only mathematical novelties, but the property could be utilized to predict protein functions with high accuracy. Biologists mostly ignored those methods, even though we went to the yeast genome database and annotated a number of unknown proteins using our method in 2002, and almost all predictions were experimentally verified.
The apathy toward fractal mathematics is not limited to the biologists however. This year, economists gave Nobel prize to Eugene Fama, whose equilibrium theory has been completely discredited using Mandelbrot’s discoveries as well as real-life observations. Warren Buffet made fun of Fama’s theory in a 1984 speech and said that his career would have been impossible, if Fama were correct. Also, Mandelbrot’s method was used successfully by Nassim Taleb’s to analyze financial market. Taleb was one of those few, who predicted 2008 financial collapse. We also predicted the financial crisis in two essays published in 2005, but using a different method.
Getting back to Barabasi’s work, a reader sent us the link to his recent paper that the readers may find interesting.
Controllability of complex networks
The ultimate proof of our understanding of natural or technological systems is reflected in our ability to control them. Although control theory offers mathematical tools for steering engineered and natural systems towards a desired state, a framework to control complex self-organized systems is lacking. Here we develop analytical tools to study the controllability of an arbitrary complex directed network, identifying the set of driver nodes with time-dependent control that can guide the system’s entire dynamics. We apply these tools to several real networks, finding that the number of driver nodes is determined mainly by the network’s degree distribution. We show that sparse inhomogeneous networks, which emerge in many real complex systems, are the most difficult to control, but that dense and homogeneous networks can be controlled using a few driver nodes. Counterintuitively, we find that in both model and real systems the driver nodes tend to avoid the high-degree nodes.
We did not follow the field closely since 2005, but would like to catch up sometime. Barabasi lab continues to be very active and posts volumes of information in their very active website. Another fascinating math we would like to ponder about is the implication of Rule 110 mentioned by Steven Wolfram. The mathematical completeness presented in Wolfram’s book seemed very helpful in describing nonlinear systems.

The Rule 110 cellular automaton (often simply Rule 110) is an elementary cellular automaton with interesting behavior on the boundary between stability and chaos. In this respect it is similar to Game of Life. Rule 110 is known to be Turing complete. This implies that, in principle, any calculation or computer program can be simulated using this automaton.
Among the 88 possible unique elementary cellular automata, Rule 110 is the only one for which Turing completeness has been proven, although proofs for several similar rules should follow as simple corollaries, for instance Rule 124, where the only directional (asymmetrical) transformation is reversed. Rule 110 is arguably the simplest known Turing complete system.[1][2]
Rule 110, like the Game of Life, exhibits what Wolfram calls “Class 4 behavior”, which is neither completely stable nor completely chaotic. Localized structures appear and interact in various complicated-looking ways.[3]
Matthew Cook proved Rule 110 capable of supporting universal computation. Rule 110 is a simple enough system to suggest that naturally occurring physical systems may also be capable of universality— meaning that many of their properties will be undecidable, and not amenable to closed-form mathematical solutions.[4]
The sooner ENCODE-like projects bankrupt the medical funding system, it is the better. Progress in science needs fewer scientists, not more.
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Kalman Filter – a Way to Reconstruct Signal from Noisy Data (as in Nanopore) [Homologus] 2013-10-28 19:53
By now, we know quite a bit about Oxford Nanopore’s experimental system from their publicly filed patent applications. The sequencer is composed of DNA molecule passing through a small pore, and the patents suggest a few possibilities for that pore-containing molecule. When the DNA passes through the pore, it produces some kind of noisy electrical signal. The challenge is in removing the noise and coming up with the actual signal. We have not seen any bioinformatics patent suggesting that the company did not come up with an elegant way to remove noise.
PacBio has shown that if someone has multiple noisy copies of long DNA sequence, it is possible to reconstruct the correct sequence at 99.99% accuracy. Some kind of Hidden Markov Model (HMM) or Bayesian algorithm is needed to do that. However, that at least requires a base-calling step, but is it possible to start from the noisy electrical sequence, skip the base calling step and go straight to error correction?
A large part of research on HMM were done by electrical engineers in 1950s and 1960s, when they tried to reconstruct communication signal passing through noisy channel. Sean Eddy made a career in bioinformatics by blissfully appropriating that research. We use the description ‘appropriation’, because he not only did not cite Viterbi in his book, but rather went to the extent of copyrighting the ‘HHMer’ name to stop electrical engineers from writing a hardware-accelerated version. Petty complaints aside, what else did the electrical engineers produce that bioinformaticians can appropriate?
As it appears, Kalman filter has not been used extensively in bioinformatics, and definitely not in topics related to reconstructing nucleotide signals. Kalman filter and extended Kalman filter became useful, because HMM was relative slow. Nanopore signal can be used as a time series to use these techniques, but we do not know what kind of difficulties will be posed by the variable speed of data arrival (i.e. DNA molecule passing through the pore at different speed).
The Kalman filter, also known as linear quadratic estimation (LQE), is an algorithm that uses a series of measurements observed over time, containing noise (random variations) and other inaccuracies, and produces estimates of unknown variables that tend to be more precise than those based on a single measurement alone. More formally, the Kalman filter operates recursively on streams of noisy input data to produce a statistically optimal estimate of the underlying system state. The filter is named for Rudolf (Rudy) E. Kálmán, one of the primary developers of its theory.
The Kalman filter has numerous applications in technology. A common application is for guidance, navigation and control of vehicles, particularly aircraft and spacecraft. Furthermore, the Kalman filter is a widely applied concept in time series analysis used in fields such as signal processing and econometrics.
The algorithm works in a two-step process. In the prediction step, the Kalman filter produces estimates of the current state variables, along with their uncertainties. Once the outcome of the next measurement (necessarily corrupted with some amount of error, including random noise) is observed, these estimates are updated using a weighted average, with more weight being given to estimates with higher certainty. Because of the algorithm’s recursive nature, it can run in real time using only the present input measurements and the previously calculated state; no additional past information is required.
From a theoretical standpoint, the main assumption of the Kalman filter is that the underlying system is a linear dynamical system and that all error terms and measurements have a Gaussian distribution (often a multivariate Gaussian distribution).
Extensions and generalizations to the method have also been developed, such as the extended Kalman filter and the unscented Kalman filter which work on nonlinear systems. The underlying model is a Bayesian model similar to a hidden Markov model but where the state space of the latent variables is continuous and where all latent and observed variables have Gaussian distributions.
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Is BWA-MEM as Good as BLASR for Aligning PacBio Reads? – Part 2 [Homologus] 2013-10-28 16:17
In an earlier commentary, we asked whether BWA-MEM is as good as BLASR for aligning PacBio reads. A number of readers, including the authors of both programs, commented here and in twitter. Below we present some preliminary results from our comparison. Please excuse us, if the following commentary appears too text-heavy. We are still in the middle of the analysis and will add a few graphs as we continue.
An Introduction to PacBio Data Format
For this comparison, we used a subset of human genomic reads released by PacBio and Eichler lab covering the genome at 10X. For those unfamiliar with PacBio data, here is a quick introduction.
PacBio sequencing is done in SMRT cells containing zero-mode waveguides (see video at the link). As a bioinformatician, all you need to know is that the data from each SMRT cell comes with an identifier like -’m120131_103014_sidney_c100278822550000001523007907041295_s1_p0′ or ‘m130610_155918_42210_c100539712550000001823089611241300_s1_p0′. The ID is likely designed by a committee and figuring out what it means is beyond your pay grade
. However, it is enough to know that the part ‘c100539702550000001823089611241314′ represents an unique identifier for the SMRT cell, and the part before that reflects the mood of the sequencer in some way.
The read libraries come in fasta, fastq or h5 format. The native format for PacBio is bax.h5, whereas fasta or fastq are derived from the h5 files. Each SMRT folder usually contains three files splitting all sequenced reads.
i) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.1.fasta,
ii) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.2.fasta,
iii) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.3.fasta.
Within each file, the reads are shown as -
>m130929_161837_42213_c100518541910000001823079209281315_s1_p0/1
AAGTTGT…….
>m130929_161837_42213_c100518541910000001823079209281315_s1_p0/2
TCTGCTGGATCGTCAAGAGGTAAAAACGCA…..
and so on. The numbers after “/” go from 1 to say 55K in file m130929_161837_42213_c100518541910000001823079209281315_s1_p0.1, ~55K to ~110K for m130929_161837_42213_c100518541910000001823079209281315_s1_p0.2 and ~110K to ~165K in m130929_161837_42213_c100518541910000001823079209281315_s1_p0.3.
As a first step of the analysis, you will need to filter and clean the raw reads and, most importantly, split them into subreads. After that preprocessing step (‘pls2fasta’ in BLASR library), you will get a second set of files, with names like -
i) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.1.subreads.fasta,
ii) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.2.subreads.fasta,
iii) m130929_161837_42213_c100518541910000001823079209281315_s1_p0.3.subreads.fasta.
The subreads FASTA file will contain IDs such as -
>m130929_161837_42213_c100518541910000001823079209281315_s1_p0/163432/27907_34462
>m130929_161837_42213_c100518541910000001823079209281315_s1_p0/163432/34510_36038
That means the sequence marked as “m130929_161837_42213_c100518541910000001823079209281315_s1_p0/163432″ in original file got split into two, and the splitting locations are given as the extra parameter on the right. Some reads from the original file will disappear at this point and some other reads will get included at full length.
An example from human libraries
For our analysis, we chose data from m130929_161837_42213_c100518541910000001823079209281315_s1_p0 SMRT cell. It is one of those 66 SMRT cell covering the human genome at 10X, and therefore likely contains 10/66 or 15% of the genome. The actual number for this SMRT cell is slightly lower – ~389M nucleotides covering the genome at 12%.
Speaking of number of reads, the original raw read files had at least 163,465 reads (we are guessing, because that file is not released). Among them, 110K reads disappeared after the pre-processing and the remaining 53,933 reads got split into 62,617 subreads. Do not worry about the great loss of 110K reads. Preprocessing step usually sees 15-20% loss of nucleotides constituting of mostly small or adapter sequences. Therefore, it is quite impressive that, with the new chemistry, PacBio achieved 500Mb/SMRT cell of throughput after pre-processing and not with the raw reads.
The N50 read size for this SMRT cell is 10,092 or ~10K after pre-processing. The longest subread has size of 41,463 nt and about 1,500 subreads are longer than 20Kb each.
BWA-MEM vs BLASR
Both Heng Li (author of BWA MEM) and Mark Chaisson (author of BLASR) commented on the applicability of BWA MEM on PacBio reads in the earlier thread. Based on our understanding of the algorithms, we believe Mark’s comment is more accurate and here is why. When someone says that PacBio reads contain 15% of errors, people tend to imagine 1 error or more in every 10 nucleotides. Mark mentioned a few months back that he often puts up two slides in his conference presentations – one with roughly 1 error in every 10 nucleotides and another with an actual PacBio read (15% error rate) – both aligned to some reference. When he asks the audience to identify the real PacBio read, they almost always select the incorrect one.
In reality, PacBio reads have longer stretches of matching segments than what our naive expectation would suggest. That is not a special feature of PacBio technology, but is an outcome of the mathematics of random numbers. Truly random coin tosses have much longer stretches of all heads or all tails than what our naive expectation of 50-50 probability distribution would suggest. BLASR paper uses this observation to find large matching seeds between the query and the reference, and then uses collection of matching seeds to generate the overall alignment.
BWA-MEM is no different except for one difference. BLASR paper by Chaisson et al. found that optimal seed size for PacBio reads would be ~16nt, whereas BWA-MEM sets the default seed size at 19nt. Apart from that, the seeding strategy of BLASR and BWA-MEM appear to be similar.
Speed, Memory, Performance
Why care about BWA-MEM, when BLASR is already available? The answer is speed. BWA-MEM is about 8-10 times as fast as BLASR. For the SMRT cell mentioned above, BWA-MEM took one hour to align the entire library, whereas BLASR is estimated to take ~8 hours (based on aligning 1/8th of the reads in one hour). For both alignments, we used default parameters.
We do not know, which algorithmic step leads to the largest difference in speed. Most likely, it is from the two-way BWT used by BWA-MEM. Please note that we ran BLASR after pre-computing the suffix array for the genome and providing it as an input in alignment step. Therefore, suffix array generation time is not included in the time difference between the algorithms.
Number of Matches for 53,933 Reads in the above file
We split the matches into four categories.
(i) BWA mem and BLASR match the read to the same chromosomal region:
46,952 reads (87%) in an estimate highly biased toward BWA MEM (see later).
(ii) BWA mem and BLASR map the read to different chromosomes:
2266 reads (4%).
(iii) BWA mem can map the read, but BLASR cannot:
3987 reads (7%).
(iv) BLASR can map the read, but BWA mem cannot:
728 reads (1.3%).
Let us take a closer look at each category to see what is going on. First group counts agreement between two algorithms, but what is ‘agreement’? We are counting all cases, where BWA MEM and BLASR map the read on the same human chromosome, but the starting location of the map may vary. The following chart shows the number of matches by varying the distances between starting locations of BWA MEM and BLASR map. The 46,952 number reported earlier allows distance of as much as 10K betweeen BWA MEM and BLASR hit (has to be same chromosome), but with 5K distance, the number of agreements is still at a respectable 46,336.
Is 5Kb distance too large to say that BWA MEM and BLASR ‘agree’? Apparently not. We went through the individual alignments, and find that often BWA MEM maps part of a read in the right location, but does not extend the match to both ends. For longer reads, BWA MEM leaves out 3Kb or more of unmapped parts on each side, whereas BLASR forces end-to-end map, as commented by Mark Chaisson. It is possible to extend the BWA MEM match by dropping the gap penalty to 0? We have seen that to work, but have not done any systematic analysis yet. A number of readers also commented in twitter BWA MEM does not go over long gaps well. What we have seen is that BWA MEM splits such matches into two separate hits, but once again, no systematic analysis has been done yet.
Second category – “BWA mem and BLASR map the read to different chromosomes”. We manually checked a few such cases and found that BWA MEM often returned the correct map identified by BLASR as its second best hit. This needs to be further investigated.
Third category – “BWA MEM can map the read, but BLASR cannot”. We have no insight into this group. It is possible that BWA MEM aligned only a small fragment (50-100 nt, possibly repeat) of the read, whereas BLASR, by forcing end-to-end match, could not see any proper agreement.
Fourth category – “BLASR can map the read, but BWA MEM cannot”. We followed up on a few such hits and found that their percentage differences with the reference, as reported by BLASR, were around 76-77%. Maybe the seeding strategy of BWA MEM fails, when the error level is too high.
We will continue to work on this topic, and report any other miracle or divine intervention that occurs to us.
The post Is BWA-MEM as Good as BLASR for Aligning PacBio Reads? – Part 2 appeared first on Homologus.
Khash by Attractive Chaos [Homologus] 2013-10-28 01:39
While trying to understand BWA and BWA MEM code, we came across a good blog site by ‘Attractive Chaos’ that the readers will find helpful.
Here is the relevant post on khash -
Speeding up khash with a power-of-2 bucket count
but in another recent commentary, the author also spent time to provide useful tips on how to parallelize code. Interestingly, we were scratching our head about the same topic, when we found the following commentary.
Parallelizing simple “for” loops
I implemented a heap-free, lock-free and wait-free scheduler for parallelizing simple independent “for” loops. For example, if we have a piece of code
data_type *data;
for (int i = 0; i < N; ++i)
do_work(data, i);where each cycle is largely independent of other cycles, we can process the loop with 4 threads:
data_type *data;
kt_for(4, do_work, data, N);The 4 threads will end at about the same time even if each cycle takes very different time to process.
The scheduler uses a simplified task stealing algorithm to balance the load of each thread. Initially, given m threads, kt_for() assigns the i-th task/cycle to thread i%m. If a thread finishes earlier than other threads, the thread will steal a task from the most loaded thread. Thus as long as there remain enough tasks, no threads will be idle.
Many other commentaries are informative. The best part - author chooses to be anonymous in this age of narcissism. It must be a bioinformatics guy from Asia.
Bookmarked !
-----------------------------
Getting back to klib and khash, what is the secret behind their performance? The author explains -
Klib is fast partly because the compiler knows the key-value type at the compile time and is able to optimize the code to the same level as type-specific code. A generic library written with void* will not get such performance boost.
Massively inserting code upon instantiation may remind us of C++'s slow compiling speed and huge binary size when STL/boost is in use. Klib is much better in this respect due to its small code size and component independency. Inserting several hundreds lines of code won't make compiling obviously slower.
The above is one reason we try to avoid STL and boost. The ease they provide in coding is often lost in performance. We are ready to take some pain in coding so that the code remains the best performing, instead of building the coolest looking Yugo.
Klib library takes all important functions to macro so that function calls turn into macro replacements to be sorted out at the compile time.
#define kv_push(type, v, x) do { \
if ((v).n == (v).m) { \
(v).m = (v).m? (v).m<<1 : 2; \
(v).a = (type*)realloc((v).a, sizeof(type) * (v).m); \
} \
(v).a[(v).n++] = (x); \
} while (0)
The post Khash by Attractive Chaos appeared first on Homologus.
Everything You Want to Know about Oxford Nanopore [Homologus] 2013-10-27 23:50
Over the last week, science blogs described the sequencing instrument of Oxford Nanopore as everything from hi-tech washing machine to Nigerian scam, ZX-81 and Turbo Pascal combined together. We emailed the CEO of this supposedly ultra-secretive company with those blog posts, and to our utter surprise, received a long email reply describing everything they accomplished over the last two years.
Well, we made up the last part in the above paragraph. No email exchange took place with their company (editing to clarify this point), but we learned a lot about their technologies. How so? Because unlike the public impression we get about them as being highly secretive, the company is actually very open and regularly posts many details about their technology online. From those postings, a knowledgeable person should be able to put together quite a bit about what they are up to and what their likely bottlenecks are. You do not need a password to access that site and simply click on the links below. The figures are not available from the linked website, but they can be easily found online without help from NSA.
1. Hairpin loop method for double strand polynucleotide sequencing using transmembrane pores
Summary of the Invention
The inventors have surprisingly demonstrated that both strands of a double stranded target polynucleotide can be sequenced by a nanopore when the two strands are linked by a bridging moiety and then separated. Furthermore, the inventors have also surprisingly shown that an enzyme, such as Phi29 DNA polymerase, is capable of separating the two strands of a double stranded polynucleotide, such as DNA, linked by a bridging moiety and controlling the movement of the resulting single stranded polynucleotide through the transmembrane pore.
The ability to sequence both strands of a double stranded polynucleotide by linking the two strands with a bridging moiety has a number of advantages, not least that both the sense and anti-sense strands of the polynucleotide can be sequenced. These advantages are discussed in more detail below.
Accordingly, the invention provides a method of sequencing a double stranded target polynucleotide, comprising:
(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety;
(b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;
(c) moving the single stranded polynucleotide through a transmembrane pore such that a proportion of the nucleotides in the single stranded polynucleotide interact with the pore; and
(d) measuring the current passing through the pore during each interaction and thereby determining or estimating the sequence of the target polynucleotide, wherein the separating in step (b) comprises contacting the construct with a
polynucleotide binding protein which separates the two strands of the target
polynucleotide.
The invention also provides:
a kit for preparing a double stranded target polynucleotide for sequencing comprising (a) a bridging moiety capable of linking the two strands of the target polynucleotide at or near one end and (b) at least one polymer;
a method of preparing a double stranded target polynucleotide for sequencing, comprising:
(a) linking the two strands of the target polynucleotide at or near one end with a bridging moiety; and
(b) attaching one polymer to one strand at the other end of the target polynucleotide and thereby forming a construct that allows the target polynucleotide to be sequenced using a transmembrane pore;
a method of sequencing a double stranded target polynucleotide, comprising:
(a) providing a construct comprising the target polynucleotide, wherein the two strands of the target polynucleotide are linked at or near one end of the target polynucleotide by a bridging moiety; (b) separating the two strands of the target polynucleotide to provide a single stranded polynucleotide comprising one strand of the target polynucleotide linked to the other strand of the target polynucleotide by the bridging moiety;
(c) synthesising a complement of the single stranded polynucleotide, such that the single stranded polynucleotide and complement form a double stranded
polynucleotide;
(d) linking the two strands of the double stranded polynucleotide at or near one end of the double stranded polynucleotide using a bridging moiety;
(e) separating the two strands of the double stranded polynucleotide to provide a further single stranded polynucleotide comprising the original single stranded polynucleotide linked to the complement by the bridging moiety;
(f) moving the complement through a transmembrane pore such that a proportion of the nucleotides in the complement interact with the pore; and
(g) measuring the current passing through the pore during each interaction and thereby determining or estimating the sequence of the target polynucleotide, wherein the separating in step (e) comprises contacting the construct with a polynucleotide binding protein which separates the two strands of the target polynucleotide;
an apparatus for sequencing a double stranded target polynucleotide, comprising: (a) a membrane; (b) a plurality of transmembrane pores in the membrane; (c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide; and (d) instructions for carrying out the method of the invention; and
an apparatus for sequencing a double stranded target polynucleotide, comprising: (a) a membrane; (b) a plurality of transmembrane pores in the membrane; and (c) a plurality of polynucleotide binding proteins which are capable of separating the two strands of the target polynucleotide, wherein the apparatus is set up to carry out the method of the invention.. Description of the Figures
Fig. 1 shows a schematic of enzyme controlled dsDNA and ssDNA translocation through a nanopore. An enzyme (e.g. Phi29 DNA polymerase) that is incubated with dsDNA having an ssDNA leader binds at the ssDNA-dsDNA interface. DNA-enzyme complexes are captured by a nanopore under an applied field. Under the field, the template strand of the DNA is slowly stripped through the enzyme in a controlled base-by-base manner, in the process unzipping the complementary primer strand of the dsDNA in or above the enzyme. Once the enzyme reaches the end of the dsDNA it falls off the D A, releasing it through the nanopore.
Fig. 2 shows another schematic of enzyme controlled dsDNA and ssDNA translocation through a nanopore. The dsDNA has a hairpin turn linking the sense and anti-sense strands of the dsDNA. Once the enzyme reaches the hairpin it remains bound to the DNA, proceeds around the hairpin turn, and along the anti-sense strand. In the hairpin and antisense regions the enzyme functions as an ssDNA molecular brake, continuing to sufficiently control translocation of the DNA through the nanopore to sequence the DNA.
Fig. 3 shows a schematic overview of reading around a hairpin of dsDNA using the ability of the enzyme to control movement in ssDNA regions. The dsDNA has a 5′-ssDNA leader to allow capture by the nanopore. This is followed by a dsDNA section, where the sense and anti-sense strands are connected by a hairpin. The hairpin can optionally contain markers (e.g. abasic residues, shown in Fig. 3 as a cross) that are observed during sequencing, which permit simple identification of the sense and anti-sense strands during sequencing. The 3 ‘-end of the anti-sense strand can optionally also have a 3′-ssDNA overhang, which if greater than ~20 bases allows full reading of the anti-sense strand (the read-head of the nanopore is -20 bases downstrand from the top of the DNA in the enzyme).
Fig. 4 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 marks the anti-sense section. This figure shows DNA sequencing of a short dsDNA construct. In this construct the dsDNA section is not connected by a hairpin, so the enzyme falls off the end of the DNA, and only the template/sense strand is sequenced (except for the last -20 bases).
Fig. 5 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. DNA sequencing of a short dsDNA construct with a hairpin. In this construct the enzyme moves along the sense strand, around the hairpin loop, and down the anti- sense strand, permitting sequencing of both the sense and the first part of the anti-sense strand.
Fig. 6 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. Similar to Fig. 5, this construct permits sequencing of both the sense and anti-sense strands, but the additional 3′-ssDNA overhang permits reading of the full length of the anti-sense strand before the enzyme falls off the end of the DNA. Fig. 7 shows a schematic of the DNA-Enzyme-nanopore complex (left) sequenced in unzipping mode through MspA nanopores using Phi29 DNA polymerase, and the consensus sequence obtained from them (right). Section 1 marks the sense section of DNA, and section 2 the anti-sense section. Similar to Fig. 5, this construct permits sequencing of both the sense and anti-sense strands, however, this construct has a single abasic residue (shown as a cross) in the hairpin, which provides a clear marker in the DNA sequence to identify the sense and anti-sense sections.
Fig. 8 shows the consensus DNA sequence of UA02 through MspA. Section 1 marks the homopolymeric 5 ‘-overhang initially in the nanopore. Section 2 marks the sense section of the DNA strand. Section 3 marks the turn. Section 4 marks the anti-sense region of the DNA strand. The polynucleotide sequence that corresponds to each section is shown below that section number.
Fig. 9 shows a schematic of a genomic template for unzipping through MspA nanopores using Phi29 DNA polymerase. It shows a general design outline for creating dsDNA suitable for reading around hairpins. The constructs have a leader sequence with optional marker (e.g. abasic DNA) for capture in the nanopore, and hairpin with optional marker, and a tail for extended reading into anti-sense strand with optional marker.
Fig. 10 shows a schematic of the adapter design for ligating ssDNA overhangs (left) and hairpin turns (right) onto genomic dsDNA. X = abasic DNA. Choi = cholesterol-TEG DNA modification.
Fig. 11 shows typical polymerase controlled DNA movement of a 400mer-hairpin through MspA using Phi29 DNA polymerase. Sense region = abasic 1 to 2. Anti-sense region = abasic 2 to 3.
Fig. 12 shows a consensus DNA sequence profile from multiple polymerase controlled DNA movements of a 400mer-hairpin through MspA. Sense region = abasic 1 to 2. Anti-sense region = abasic 2 to 3.
Fig. 13 shows a schematic of an alternative sample preparation for sequencing. A construct is illustrated comprising the target polynucleotide and a bridging moiety (hairpin) linking the two strands of the target polynucleotide. The construct also comprises a leader polymer (a single stranded sequence), a tail polymer (also a single stranded sequence) and an abasic marker region within the leader. The marker may prevent the enzyme from making the template completely blunt ended i.e. filling in opposite the required leader ssDNA. A strand displacing polymerase (nucleic acid binding protein) separates the two strands of the construct, initiating either via a complementary primer or by protein primed amplification from the tail polymer. A complement is generated to the resulting single stranded polynucleotide. The complement and the original sense and antisense single stranded polynucleotide analyte can be further modified by addition of a second bridging moiety (hairpin).
Fig. 14 shows a specific preparation of the construct comprising the target
polynucleotide.
Fig. 15 shows where amplification may be added as part of the sample preparation to aid the detection of epigenetic information. A nucleotide has been constructed so that the following information is read through the pore: sense (original), antisense (original), bridging moiety, sense (replicate), antisense (replicate). Information on the methylated base (mC) is therefore obtained four times.
Fig. 16 shows how RNA can be sequenced. A bridging moiety is attached to a piece of
R A and the DNA reverse complement added to the RNA via a reverse transcriptase. The RNA is read, followed by the DNA of the reverse complement.
Fig. 17 shows a schematic of helicase controlled dsDNA and ssDNA translocation through a nanopore. The dsDNA has a hairpin turn linking the sense and anti-sense strands of the dsDNA. Once the enzyme reaches the hairpin it remains bound to the DNA, proceeds around the hairpin turn, and along the anti-sense strand. In the hairpin and antisense regions the enzyme functions as an ssDNA molecular brake, continuing to sufficiently control translocation of the DNA through the nanopore to sequence the DNA.
Fig. 18 shows the polynucleotide MONO hairpin construct (SEQ ID NOs: 29 to 35) used in Example 4.
Fig. 19 shows a typical helicase controlled DNA movement of a 400 bp hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S-G77S- L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). Sense = region 1. Anti-sense = region 2.
Fig. 20 shows the beginning of a typical helicase controlled DNA movement of a 400 bp hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations
G75S/G77S/L88N/Q126R)). The polyT region at the beginning of the sequence is highlighted with a * and the abasic DNA bases as a #.
Fig. 21 shows the transition between the sense and antisense regions of a typical helicase controlled DNA movement of a 400 bp-hairpin (SEQ ID NOs: 29 to 35 connected as shown in Fig. 18) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). The transition region between the sense and antisense regions of the sequence is highlighted by a * , the sense region labeled 1 and the antisense region labeled 2. Fig. 22 shows an example sample prep method for forming DUO hairpin constructs. The double stranded DNA analyte is contacted by and modified to contain a Y-shaped adapter (the sense strand (SEQ ID NO: 29 attached to SEQ ID NO: 30 via four abasic DNA bases) of this adaptor contains the 5′ leader, a sequence that is complementary to the tether (SEQ ID NO: 35, which at the 3′ end of the sequence has six iSpl 8 spacers attached to two thymine residues and a 3′ cholesterol TEG) and 4 abasics and the antisense half of the adaptor contains a 3′ hairpin (SEQ ID NO: 31)) at one end of the duplex and a hairpin (SEQ ID NO: 32) at the other. The Y- shaped adapter itself also carries a 3 ‘-hairpin (SEQ ID NO: 31), which allows extension either by a polymerase or by ligation. This extension is preferentially carried out by a mesophilic polymerase that has strand displacement activity. As the polymerase extends from the 3 ‘ of the Y-shaped adapter hairpin (SEQ ID NO: 31) it copies the antisense strand (SEQ ID NO: 34) and so displaces the original sense strand (SEQ ID NO: 33). When the polymerase reaches the end of the antisense strand (SEQ ID NO: 34) it fills-in opposite the hairpin (SEQ ID NO: 32) and then begins to fill-in opposite the now single stranded and original sense strand (SEQ ID NO: 33). Extension is then halted by a section of abasic or spacer modifications (other possible modifications which could halt enzyme extension include RNA, PNA or morpholino bases and iso-dC or iso-dG) to leave the 5 ‘-region of the Y-shaped adapter single stranded (SEQ ID NO: 29).
Fig. 23 shows the specific preparation method used in Example 5 for preparing a DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25). A -400 bp region of PhiX 174 was PCR amplified with primers containing Sad and Kpnl restriction sites (SEQ ID Nos: 27 and 28 respectively). Purified PCR product was then Sad and Kpnl digested before aY- shaped adapter (sense strand sequence (SEQ ID NO: 29 attached to SEQ ID NO: 30 via four abasic DNA bases) is ligated onto the 5′ end of SEQ ID NO: 33 and the anti-sense strand (SEQ ID NO: 31) is ligated onto the 3 ‘ end of the SEQ ID NO: 34) and a hairpin (SEQ ID NO: 32, used to join SEQ ID NO’s: 33 and 34) were ligated to either end, using T4 DNA ligase (See Fig. 18 for final DNA construct). The doubly ligated product was PAGE purified before addition of lenow DNA polymerase, SSB and nucleotides to allow extension from the Y-shaped adapter hairpin (SEQ ID NO: 31). To screen for successful DUO product a series of mismatch restriction sites were incorporated into the adapter sequences, whereby the enzyme will cut the analyte only if the restriction site has been successfully replicated by the DUO extension process.
Fig. 24 shows that the adapter modified analyte (MONO, SEQ ID NOs: 29-35 connected as shown in Fog. 18) in the absence of polymerase does not digest with the restriction enzymes (see gel on the left, Key: M = Mfel, A = Agel, X = Xmal, N = NgoMIV, B = BspEl), due to the fact they are mismatched to one another. However, on incubation with polymerase there is a noticeable size shift and the shifted product (DUO, SEQ ID NOs: 29-36 connected as shown in Fig. 25) now digests as expected with each of the restriction enzymes (see gel on the right, Key: M = Mfel, A = Agel, X = Xma\, N = NgoMIV, B = BspEl).
Fig. 25 shows the polynucleotide DUO hairpin construct (SEQ ID NOs: 29 to 36) used in Examples 6.
Fig. 26 shows two typical helicase controlled DNA movements for the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations
G75S/G77S/L88N/Q126R)). Sense original = region 1. Anti-sense original = region 2. Sense replicate = region 3. Anti-sense replicate = region 4.
Fig. 27 shows an expanded view of a typical helicase controlled DNA movement for the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations G75S/G77S/L88N/Q126R)). Sense original = region 1. Anti-sense original = region 2. Sense replicate = region 3. Anti-sense replicate = region 4.
Fig. 28 shows an expanded view of a typical transition between the sense original and antisense original regions of the DUO hairpin construct (SEQ ID NOs: 29 to 36 connected as shown in Fig. 25) when under helicase controlled DNA movement through an MspA nanopore (MS(B1-G75S-G77S-L88N-Q126R)8 MspA (SEQ ID NO: 2 with the mutations.
An apparatus for supporting an array of layers of amphiphilic molecules, the apparatus comprising: a body,formed in a surface of the body, an array of sensor wells capable of supporting a layer of amphiphilic molecules across the sensor wells, the sensor wells each containing an electrode for connection to an electrical circuit, and formed in the surface of the body between the sensor wells, flow control wells capable of smoothing the flow of a fluid across the surface.
The invention relates to a new method of determining in a sample the presence or absence of one or more analyte members of a group of two or more analytes. The invention therefore relates to a multiplex assay for determining the presence or absence of each analyte in a group of multiple analytes. The assay uses aptamers and transmembrane pores.
4. Analysis of measurements of a polymer
A time-ordered series of measurements of a polymer made during translocation of the polymer through a nanopore are analysed. The measurements are dependent on the identity of k-mers in the nanopore, a k-mer being k polymer units of the polymer, where k is a positive integer. The method involves deriving, from the series of measurements, a feature vector of time-ordered features representing characteristics of the measurements; and determining similarity between the derived feature vector and at least one other feature vector.
5. Method for characterising a polynucelotide by using a xpd helicase
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and an XPD helicase. The helicase controls the movement of the target polynucleotide through the pore.
The invention relates to a new method of characterising a target polynucleotide. The method uses a pore and a Hel308 helicase or amolecular motor which is capable of binding to the target polynucleotide at an internal nucleotide. The helicase or molecular motor controls the movement of the target polynucleotide through the pore.
7. Analysis of a polymer comprising polymer units
A sequence of polymer units in a polymer (3), eg. DNA, is estimated from at least one series of measurements related to the polymer, eg. ion current as a function of translocation through a nanopore (1), wherein the value of each measurement is dependent on a k-mer being a group of k polymer units (4). A probabilistic model, especially a hidden Markov model (HMM), is provided, comprising, for a set of possible k-mers: transition weightings representing the chances of transitions from origin k-mers to destination k-mers; and emission weightings in respect of each k-mer that represent the chances of observing given values of measurements for that k-mer. The series of measurements is analysed using an analytical technique, eg. Viterbi decoding, that refers to the model and estimates at least one estimated sequence of polymer units in the polymer based on the likelihood predicted by the model of the series of measurements being produced by sequences of polymer units. In a further embodiment, different voltages are applied across the nanopore during translocation in order to improve the resolution of polymer units.
8. Piston seal
A piston head for a syringe pump comprises a barrier portion for driving fluid through a syringe pump barrel, wherein a peripheral section of the barrier portion is shaped to seal against the syringe pump barrel; and a resilient member arranged to resist deformation of the shaped peripheral section of the barrier portion.
How about data? We have not seen any nucleotide-level data yet, but the first patent mentioned above contains many figures showing their electrical signals. One informative comment at OmicsOmics blog speculated on what could be going on at the company.
Interesting discussion here. I too went ahead and glanced through OxNano’s patent applications. Based on my very quick review, here is what I found
1. They have done extensive amount of work on mspA, but Illumina licenced the pore now. Not sure what is going on there
2. The closest I saw anything related to sequencing was a bioinformatics patent application that describes how to convert K mer signatures to sequence – there was’t any sequencing data in that application
3. They seem to have some cool motors. Helicases from some extremophiles
4. One new pore called lysenin, not sure how it compares to mspA. No sequencing information in the patent application
5. Some algorithms to build consensus among traces (squiggles, if that is the terminology we are using)
6. There was one application where the did some algorithmic learning on a known piece of DNA, I didn’t see that extended to an unknown sampleRegarding the comment that investors probably saw sequencing data, that could be true. Wonder if that caused the rift with Illumina. They clearly convinced Illumina with a sequencing scheme, now they have convinced other investors of a different scheme. Time to convince the users.
Our views are in line with another comment -
The squiggles have been around for quite some time now. Both the Akeson and Gundlach labs have published the squiggles. Credit to Oxford for putting things in a nice package, but unless I see them convert the squiggles to sequence, I, sadly, will have to assume they are being evasive and just trying to buy time.
…
This other thought, I keep having because I hear how secretive and paranoid Oxford are. I am beginning to wonder if it is just a rouse to hide serious deficiencies they are trying, or unable to fix (with lots of investors and the world looking at them).
Buy time is what it is.
The post Everything You Want to Know about Oxford Nanopore appeared first on Homologus.
Markdown or LaTeX? [Yihui Xie] 2013-10-19 03:00
What happens if you ask for too much power from Markdown?

R Markdown is one of the document formats that knitr supports, and it is probably the most popular one. I have been asked many times about the choice between Markdown and LaTeX, so I think I'd better wrap up my opinions in a blog post. These two languages (do you really call Markdown a language?) are kind of at the two extremes: Markdown is super easy to learn and type, but it is primarily targeted at HTML pages, and you do not have fine control over typesetting ( really? really?), because you only have a very limited number of HTML tags in the output; LaTeX is relatively difficult to learn and type, but it allows you to do precise typesetting (you have control over anything, and that is probably why a lot of time can be wasted).
What is the root problem? I think one word answers everything: page! Why do we need pages? Printing is the answer.
In my eyes, the biggest challenge for typesetting is to arrange elements properly with the restriction of pages. This restriction seems trivial, but it is really the root of all "evil". Without having to put things on pages, life can be much easier in writing.
What is the root of this root problem in LaTeX? One concept: floating environments. If everything comes in a strictly linear fashion, writing will be just writing; typesetting should be no big deal. Because a graph cannot be broken over two pages, it is hard to find a place to put it. By default, it can float to unexpected places. The same problem can happen to tables (see the end of a previous post). You may have to add or delete some words to make sure they float to proper places. That is endless trouble in LaTeX.
There is no such a problem in HTML/Markdown, because there is no page. You just keep writing, and everything appears linearly.
There is no fault being greedy, and it is natural to ask the question whether one can have both HTML and PDF output from a single source document. The answer is maybe yes: you can go from LaTeX to HTML, or from Markdown to LaTeX/PDF.
But remember, Markdown was designed for HTML, and LaTeX was for PDF and related output formats. If you ask for more power from either language, the result is not likely be ideal, otherwise one of them must die.
If your writing does not involve complicated typesetting and primarily consists of text (especially no floating environments), go with Markdown. I cannot think of a reason why you must use LaTeX to write a novel. See Hadley's new book Advanced R programming for an excellent example of Markdown + knitr + other tools: the typesetting elements in this book are very simple -- section headers, paragraphs, and code/output. That is pretty much it. Eventually it should be relatively easy to convert those Markdown files to LaTeX via Pandoc, and publish a PDF using the LaTeX class from Chapman & Hall.
For the rest of you, what I'd recommend is to think early and make a decision in the beginning; avoid having both HTML and PDF in mind. Ask yourself only one question: must I print the results nicely on paper? If the answer is yes, go with LaTeX; otherwise just choose whatever makes you comfortable. The book Text Analysis with R authored by Matthew Jockers is an example of LaTeX + knitr. Matt also asked me this question about Markdown vs LaTeX last week while he was here at Iowa State. For this particular book, I think Markdown is probably OK, although I'm not quite sure about a few environments in the book, such as the chapter abstracts.
It is not obvious whether we must print certain things. I think we are just too used to printing. For example, dear professors, must we print our homework? (apparently Jenny does not think so; I saw her grade homework on RPubs.com!) Or dear customers, must we submit reports in PDF? ... In this era, you have laptops, iPad, Kindle, tablets and all kinds of electronic devices that can show rich media, why must you print everything (in black and white)?
For those who are still reading this post, let me finish with a side story: Matt, a LaTeX novice, taught himself LaTeX a few months ago, and he has finished the draft of a book with LaTeX! Why are you still hesitating about the choice of tools? Shouldn't you just go ahead and get the * done? Although all roads lead to Rome, some people die at the starting line instead of on the roads.
After Three Months I Cannot Reproduce My Own Book [Yihui Xie] 2013-09-05 03:00
I thought I could easily jump to a high standard (reproducibility), but I failed.

Some of you may have noticed that the knitr book is finally out. Amazon is offering a good price at the moment, so if you are interested, you'd better hurry up.
I avoided the phrase "Reproducible Research" in the book title, because I
did not want to take that responsibility, although it is related to
reproducible research in some sense. The book was written with knitr v1.3
and R 3.0.1, as you can see from my sessionInfo() in the preface.
Three months later, several things have changed, and I could not reproduce the book, but that did not surprise me. I'll explain the details later. Here I have extracted the first three chapters, and released the corresponding source files in the knitr-book repository on Github. You can also find the link to download the PDF there. This repository may be useful to those who plan to write a book using R.
What I could not reproduce were not really important. The major change in
the recent knitr versions was the syntax highlighting commands, e.g.
\hlcomment{} is \hlcom{} now, and the syntax highlighting has been
improved by the highr package
(sorry, Romain). This change brought a fair amount of changes when I look at
git diff, but these are only cosmetic changes.
I tried my best to avoid writing anything that is likely to change in the future into the book, but as a naive programmer, I have to say sorry that I have broken two little features, although they may not really affect you:
error = FALSE instead of the package option stop_on_error,
which has been deprecated (Section 6.2.4);## ---- instead of ## @knitr now;Actually the backward-compatibility is still there, so they will not really break until a long time later.
With exactly the same software environment, I think I can reproduce the book, but that does not make much sense. Things are always evolving. Then there are two types of reproducible research:
I think the latter is more valuable. Being reproducible alone is not the goal, because you may be reproducing either real findings or simply old mistakes. As Roger Peng wrote,
[...] reproducibility cannot really address the validity of a scientific claim as well as replication
Roger's recent three blog posts on reproducible research are very worth reading. This blog post of mine is actually not quite relevant (no data analysis here), so I recommend my readers to move over there after you haved checked out the knitr-book repository.
My first Bioconductor conference (2013) [Yihui Xie] 2013-07-21 03:00
The BioC 2013 conference was held from July 17 to 19. I attended this conference for my first time, mainly because I'm working at the Fred Hutchinson Cancer Research Center this summer, and the conference venue was just downstairs! No flights, no hotels, no transportation, yeah.
Last time I wrote about my first ENAR experience, and let me tell you why the BioC conference organizers are smart in my eyes.
I do not need to explain this simple design -- it just will not flip to the damn blank side:

The program book was only four pages of the schedule (titles and speakers). The abstracts are online. Trees saved.
There were plenty of lightning talks. You can talk whatever you want.
On the developer's day, Martin Morgan presented some buggy R code to the audience (provided by Laurent Gatto), and asked us to debug it right there. Wow!
The registration includes almost everything: lunch, beer, wine, coffee, fruits, snacks, and most importantly, Amazon Machine Instances (AMI)!
This is a really shiny point of BioC! If you have ever tried to do a software tutorial, you probably know the pain of setting up the environment for your audience, because they use different operating systems, different versions of packages, and who knows what is going to happen after you are on your third slide. At a workshop last year, I had the experience of spending five minutes figuring out why a keyboard shortcut did not work for one Canadian lady in the audience, and it turned out she was using the French keyboard layout.
The BioC organizers solved this problem beautifully by installing the RStudio server on AMI. Every participant was sent a link to the Amazon virtual machine, and all they need is a web browser and wireless connection in the room. All people run R in exactly the same environment.
Isn't that smart?
I do not really know much about biology, although a few biological terms have been added to my volcabulary this summer. When a talk becomes biologically oriented, I will have to give up.
Simon Urbanek talked about big data in R this year, which is unusual, as mentioned by himself. Normally he shows fancy graphics (e.g. iplots). I did not realize the significance of this R 3.0.0 news item until his talk:
It is now possible to write custom connection implementations outside core R using
R_ext/Connections.h. Please note that the implementation of connections is still considered internal and may change in the future (see the above file for details).
Given this new feature, he implemented the HDFS connections and 0MQ-based connections in R single-handedly (well, that is always his style).
You probably have noticed the previous links are Github repositories. Yes! Some R core members really appreciate the value of social coding now! I'm sure Simon does. I'm aware of other R core members using Github quietly (DB, SF, MM, PM, DS, DTL, DM), but I do not really know their attitude toward it.
Joe Cheng's Shiny talk is shiny as usual. Each time I attend his talk, he will show a brand new amazing demo. Joe is the only R programmer that makes me feel "the sky is the limit (of R)". The audience were shocked when they saw a heatmap that they were so familiar with suddently became interactive in a Shiny app! BTW, Joe has a special sense of humor when he talks about an area in which he is not an expert (statistics or biology).
RStudio 0.98 is going to be awesome. I'm not going to provide the links here, since it is not released yet. I'm sure you will find the preview version if you really want it.
Given Biocondutor's open-mindedness to new technologies (GIT, Github, AMI, Shiny, ...), let's see if it is going to take over the world. Just kidding. But not completely kidding. I will keep the conversation going before I leave Seattle around mid-August, and get something done hopefully.
If you have any feature requests or suggestions to Bioconductor, I will be happy to serve as the "conductor" temporarily. I guess they should set up a blog at some point.
R Package Versioning [Yihui Xie] 2013-06-27 03:00
This should be what it feels like to bump the major version of your software:

For me, the main reason for package versioning is to indicate the (slight or significant) differences among different versions of the same package, otherwise we can keep on releasing the version 1.0.
That seems to be a very obvious fact, so here are my own versioning rules, with some ideas borrowed from Semantic Versioning:
major.minor.patch (x.y.z), e.g., 0.1.7x.y is released to CRANx.y.z is always the development version, and each time a new feature or
a bug fix or a change is introduced, bump the patch version, e.g., from
0.1.3 to 0.1.40.1 to 0.20.18 to 1.01.0 does not imply maturity; it is just because it is
potentially very different from 0.x (such as API changes); same thing
applies to 2.0 vs 1.0I learned the rule #3 from Michael Lawrence (author of RGtk2) and I
think it is a good idea. In particular, it is important for brave users who
dare install the development versions. When you ask them for their
sessionInfo(), you will be aware of which stage they are at.
Rule #2 saves us a little bit energy in the sense that we do not need to write or talk about the foo package 1.3.548, which is boring to type or speak. Normally we say foo 1.3. As a person whose first language is not English, speaking the patch version does consume my brain memory and slows down my thinking while I'm talking. When I say it in Chinese, I feel boring and unnecessarily geeky. Yes, I know I always have weird opinions.
You Do Not Need to Tell Me I Have A Typo in My Documentation [Yihui Xie] 2013-06-10 03:00

So I just got yet yet another comment saying "you have a typo in your documentation". While I do appreciate these kind reminders, I think it might be a good exercise for those who want to try GIT and Github pull requests, which make it possible for you to contribute to open source and fix obvious problems with no questions being asked -- just do it yourself, and send the changes to the original author(s) through Github.
The official documentation for Github pull requests is a little bit verbose for beginners. Basically what you need to do for simple tasks are:
Fork button and clone the repository in your own account;Pull Request button to send a request to the original author;For trivial changes, sometimes I accept them on my cell phone while I'm still in bed. No extra communication is needed.
Occasionally I see reports of this kind of trivial documentation changes in the R-devel mailing list, and I believe that is just horribly inefficient. You could have done this quietly and quickly, and the developers could have merged the changes with a single mouse click. (Oh, okay, well, you know, SVN, mailing lists, ...)
For the knitr repository, it has two
branches: master and gh-pages. The R package lives in the master
branch, and the knitr website lives in the gh-pages branch. If you
want to fix any problems in the website, just check out the gh-pages:
git checkout gh-pages
All pages were written in Markdown, so edit them with your favorite text
editor. For example, as the above comment pointed out, I omitted a right
parenthesis ) in _posts/2012-02-24-sweave.md, and you just add it, save
the file, write a GIT commit message, push to your repository and send the
pull request.
I know I can do this by myself in five seconds, and it takes me way more time to write this blog post, but I just want everybody to know how people with different skill levels can play their roles in software development.
Let's see how many minutes it takes for the pull request to come after I publish this blog post. Hurry!! :)
A Few Tips for Writing an R Book [Yihui Xie] 2013-06-03 03:00
I just finished fixing (hopefully all) the problems in the knitr book returned from the copy editor. David Smith has kindly announced this book before I do. I do not have much to say about this book: almost everything in the book can be found in the online documentation, questions & answers and the source code. The point of buying this book is perhaps you do not have time to read through all the two thousand questions and answers online, and I did that for you.
This is my first book, and obviously there have been a lot for me to learn about writing a book. In retrospect, I want to share a few tips that I found useful (in particular, for those who plan to write for Chapman & Hall):
knitr made it a lot easier to manage R code and its output for the book; for example, I could quickly adapt to R 3.0.1 from 2.15.3 after I came back from a vacation; if I were to write a second edition, I do not think I will have big trouble with my R code in the book (it is easy to make sure the output is up-to-date);fivenum() in base R was changed from R 2.15.3 to 3.0.0 thanks to GIT (R core have been updating base R everywhere!);krantz.cls (by Chapman & Hall):
\renewcommand{\textfraction}{0.05}
\renewcommand{\topfraction}{0.8}
\renewcommand{\bottomfraction}{0.8}
\renewcommand{\floatpagefraction}{0.75}
I'm aware of the float package and the H option, and options like !tbp; I just do not want to force LaTeX to do anything. It may or may not be happy at some point.
\usepackage{emptypage} in the preamble to make empty pages really empty, as required by the copy editor.krantz.cls does not work with the hyperref package, meaning that you cannot create bookmarks in the PDF; I have posted the solution here.when you want to use which, use that instead, unless there is a comma ahead, or you really want to emphasize a very specific object; e.g.,
"here is a package that is helpful" (correct)
"here is a package which is helpful" (wrong)
"we will introduce an extremely important technology next, which has revolutionized the life of poor statisticians"
it is "A, B, and C" instead of "A, B and C"
cairo_pdf() device when possible; in knitr, this means the chunk option dev = 'cairo_pdf'; the reason for the choice of cairo_pdf() over the normal pdf() device is that it can embed fonts in the PDF plot files, otherwise the copy editor will require you to embed all the fonts in the final PDF file of the book; normally pdflatex will embed fonts, and if there are fonts that are not embedded, it is very likely that they are from R graphics;pdftk, because you may want to use it finally, e.g., replace one page with a blank page in the frontmatter;One thing I did not really understand was the punctuation marks like commas and periods should go inside quotation marks, e.g.,
I have "foo" and "bar."
This makes me feel weird. I'm more comfortable with
I have "foo" and "bar".
There was also one thing that I did not catch by version control -- one figure file went wrong and I did not realize it, because normally I do not put binary files under version control. Fortunately, I caught it by my eyes. Karl Broman mentioned the same problem to me a while ago. I know there are tools for comparing images (ImageMagick, for example), and I was just too lazy to learn them.
I will be glad to know the experience of other authors, and will try to update this post according to the comments.
Travis CI for R! (not yet) [Yihui Xie] 2013-04-12 03:00
A few days ago I wrote about Travis CI, and was wondering if we could integrate the testing of R packages into this wonderful platform. A reader (Vincent Arel-Bundock) pointed out in the comments that Travis was running Ubuntu that allows you to install software packages at your will.
I took a look at the documentation, and realized they were building and testing packages in virtual machines. No wonder sudo apt-get works. Remember apt-get -h | tail -n1:
This APT has Super Cow Powers. (APT有超级牛力)
Now we are essentially system admins, and we can install anything from Ubuntu repositories, so it does not really matter that Travis CI does not support R yet. Below are a few steps to integrate your R package (on Github) into this system:
.travis.yml;.travis.yml for the knitr package if you want, or write your own;
~/R to install add-on R packages so that I do not have to type sudo everywhereR CMD check requires all packages in Suggests as well, I install knitr using install.packages(dep = TRUE) to make sure all relevant packages are installedmake install and make check are wrappers of R CMD build and R CMD check respectively, defined in the Makefile.travis.yml to Github, and Travis CI will start building your package when a worker is available (normally within a few seconds);By default you will receive email notifications when there are changes in the build. You can also find the guide on the build status image in the documentation as well, e.g. 
What I described here actually applies to any software packages (not only R), as long as the dependencies are available under Ubuntu, or you know how to build them.
OK, it works, but we are still a little bit far from what CRAN does, because Travis CI does not have official support for R. Each time we have to install one Gigabyte of additional software to create the R testing environment (sigh, if only R did not have to tie itself to LaTeX). If these packages are pre-built in the virtual machines, it will save us a lot of time.
The second problem is, there is no Windows support on Travis CI (one developer told us on Twitter that it was coming). There is a page for OS X, but I did not really figure out how to build software under OS X there.
The third problem is Travis CI only builds and tests packages; it does not provide downloads like CRAN. Perhaps we can upload the packages using encryption keys to our own servers.
I will shut up here since I realized I was not being constructive. Let me spend more time thinking about this, and I love to hear suggestions from readers as well.
So, two potential Google Summer of Code projects:
Travis CI for R? [Yihui Xie] 2013-04-07 03:00
I'm always worried about CRAN: a system maintained by FTP and emails from real humans (basically one of Uwe, Kurt or Prof Ripley). I'm worried for two reasons:
I have a good solution for 2, which is to keep silent when your submission passes the check system, and say "Sorry!" no matter if you agree with the reason or not when it did not pass (which made one maintainer unhappy), but do not argue -- just go back and fix the problem if you know what is the problem; or use dark voodoo to hide (yes, hide, not solve) the problem if you are sure you are right. If you read the mailing list frequently, you probably remember that if (CRAN) discussion. The solution in my mind was if (Sys.getenv('USER') == 'ripley').
The key is, do not argue. Silence is gold.

The CRAN maintainers have been volunteering their time, and we should respect them. The question is, will this approach scale well with the growth of packages? Or who should be in charge of R CMD check?
We, the poor authors, cannot guaranttee that every time our packages can pass CRAN's machines due to all kinds of reasons. Some problems are actually easy to fix without a real human yelling at us. On the other hand, if the package fortunately passes R CMD check, we do not really need an email from a real human acknowledging "thanks, on CRAN now".
Travis CI is an excellent platform for continuous integration of software packages. You do not need to interact with a real person by email -- each time you push to Github, your package will be automatically built and checked. If there are problems, you will be notified automatically.
A similar platform in the R world is Bioconductor. It has the best two components in software development: version control (although sadly SVN) and continuous checking. I do not know if CRAN will catch up one day. I'm not very optimistic about it; perhaps a more realistic approach is to start a Google Summer of Code project on introducing R into Travis CI. I have no idea how difficult that will be, but I will definitely be thrilled if it comes true this year.
Anyone?
Update on 04/16/2013: just to clarify, what Bioconductor does is not strictly continuous integration (yet) in the sense that it builds packages daily instead of immediately on changes.
On ENAR, or Statistical Meetings in General [Yihui Xie] 2013-03-14 03:00
Last year I accepted an invitation from Ben to go to ENAR 2013 -- my first ENAR. I used to go to JSM and useR!, and apparently I enjoy useR! most. The reason is not, or not only, because I'm more of a technical person. It is just hard to concentrate at large statistical conferences. I want to make a few suggestions from the perspective of a student, although it is unlikely that any future conference chairs will come here and listen to me:
There's a world outside #ENAR2013 but barbed wire to prevent us from getting there. twitter.com/kwbroman/statu…
— Karl Broman (@kwbroman) March 12, 2013
By comparison, useR! conferences often take place in a university campus. Last year it was in Vanderbilt, and they provided dorms to students. I lived happily in the dorm, because all I wanted was a place to sleep (there was free wireless too); nothing luxury. Usually there are also inexpensive restaurants on campus. I met helpful local students/researchers there who gave me free ride to some scenic spots, and I had to fight to pay my tickets by myself (but was still treated). It was just a touching trip and I managed to make acquaintance quite a few interesting people (Frank Harrell, Bill Venables and Kevin Coombes, etc).
ENAR "kindly" included a ticket for the Epcot theme park in the registration fee. What did I do in the theme park? I had a dinner in an expensive restaurant (Karl felt guilty for taking me there and generously treated me), and watched a 10-minute fireworks show. Yes, we were so nerdy that we kept on discussing the role of measure theory and Github in a place where we were supposed to say hello to the Mickey mouse.
At #Epcot with @mstephens999 and @xieyihui for #ENAR2013 twitter.com/kwbroman/statu…
— Karl Broman (@kwbroman) March 13, 2013
So my major suggestion to the big statistical meetings is, create an environment which emphasizes the communication among people and do not include distracting activities in the registration package by default. There are a couple of small things you can do, for example:
Okay, rants ended. Positive energy coming.
As I said on Twitter, I was happy to meet Karl Broman (who introduced Matthew Stephens to me later, the smartest person in statistics and human genetics according to Karl) and John Muschelli there. I noticed Karl long time ago, mainly due to Top ten worst graphs, but have never met him.
I did not know much about the Johns Hopkins biostat department before I visited them last year, and it has become a place that surprises me more and more. It is a weird and crazy department. I like people for bizarre reasons. For instance, I like Jeff Leek because he prefers steak to be well done (me too). Rafa hides jokes under his very serious-looking face. "Behind the Tan Door" is the best video in the history of statistics. Karl has a series of hilarious stories about the JHSPH logo. There are people in the world that you know for sure you will be excited to meet. Currently I have yet another person on my list: Tyler Rinker.
In case you have not seen it, I strongly recommend you read (and apply for) the postdoctoral fellow position in reproducible research at Hopkins. Note in particular the phrase "serious moxie"!!
So do I regret ENAR? Certainly no. If I can organize my time more efficiently meeting more people like those above, it will be even better. BTW, if you are not the 20 people in my session, feel free to check out my slides on knitr (Brian Bot simply called them "animated gifs" instead of "slides").
Contribute to The R Journal with LyX/knitr [Yihui Xie] 2013-02-17 03:00
(This paragraph is pure rant; feel free to skip it) I have been looking forward to the one-column LaTeX style of The R Journal, and it has arrived eventually. Last time I mentioned "it does not make sense to sell the cooked shrimps"; actually there is another thing that does not make sense in my eyes, which is the two-column LaTeX style. I just hate it. Two-column may save a little bit space in typesetting compared to one-column, but it brings huge inconvenience to the readers who do not have a big enough screen. For each single page, you have to scroll down to read the left column, scroll back and up to read the right column, then scroll down... So you just scroll up and down, up and down, ... until you are bored by this PITA.

I have ported the new RJournal.sty to LyX, and you can find the relevant files in my lyx repository. To write articles in LyX with knitr, check out or download the repository and follow these steps:
User directory from the LyX menu: Help --> Aboutlayouts folder to your user directory;RJournal.sty from the R Journal website and put it in your texmf tree so that LaTeX can find it (this might be the most challenging step if you do not know enough about LaTeX, and I do not want to explain this painful topic);PATH (again this is a painful topic that I hate to explain) and install.packages('knitr') in R;Tools --> Reconfigure and restart LyX;Now you should be able to open templates/RJournal.lyx and compile it. I have made a quick video of the process below:
So you have no execuse to escape reproducible research! It is even easier than writing in Word to contribute a reproducible article to The R Journal now.
P.S. I will try to submit this new layout file RJournal.layout as well as the template RJournal.lyx to the LyX development team if I do not hear any problems from users.
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